DNA encoding high-affinity melatonin receptors

ABSTRACT

Disclosed are cDNAs and DNAs encoding high-affinity melatonin 1a and 1b receptors and the recombinant polypeptides expressed from such cDNAs. The recombinant receptor polypeptides, receptor fragments and analogs expressed on the surface of cells are used in methods of screening candidate compounds for their ability to act as agonists or antagonists to the effects of interaction between melatonin and high-affinity melatonin receptor. Agonists are used as therapeutics to reentrain endogenous melatonin rhythms as a means of treating circadian rhythm disorders in humans and control reproductive cycles in seasonally breeding animals. Antagonists are used as therapeutics to control the initiation or timing of puberty in humans. Antibodies specific for a high-affinity melatonin receptor (or receptor fragment or analog) and their use as a therapeutic are also disclosed.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made at least in part with funds from the Federalgovernment, and the government therefore has rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of our earlier filed U.S.application Ser. No. 08/319,887 filed Oct. 7, 1994 now abandoned, whichapplication is a continuation-in-part of our earlier filed U.S.application Ser. No. 08/261,857 filed Jun. 17, 1994 now abandoned, whichapplication is incorporated herein by reference in its entirety and towhich application we claim priority under 35 USC §120.

BACKGROUND OF THE INVENTION

The invention relates to nucleic acids and their encoded high-affinitymelatonin receptor proteins.

The high-affinity melatonin receptor is a membrane protein that iscoupled to guanine nucleotide binding proteins (G proteins). G proteins,in turn, communicate ligand-activated receptor signals to theappropriate intracellular effector system(s). The hormone, melatonin,inhibits adenylyl cyclase causing a decrease in intracellular cyclic AMP(cAMP) concentration.

Melatonin, the principal hormone of the vertebrate pineal gland, elicitspotent neurobiological effects. Melatonin influences circadian rhythmand mediates the effects of photoperiod on reproductive function inseasonally breeding mammals. In humans, melatonin administration hasbeen shown to alleviate the symptoms of jet lag after air travel acrossseveral time zones. The hormone also has potent sedative effects inhumans and may be a useful hypnotic agent.

Melatonin exerts its photoperiodic and circadian effects throughpharmacologically specific, high-affinity receptors (Dubocovich, M. L.and Takahashi, J., P.N.A.S. USA (1987) 84:3916-3920; Vanecek, J., J.Neurochem. (1988) 51:1436-1440; Reppert et al., (1988) supra). Inseasonally breeding mammals, pineal melatonin secretion regulatesseasonal responses to changes in day length (Bartness, T. J. andGoldman, B. D., Experientia (1989) 45:939-945; Karsch et al., RecentProg. Horm. Res. (1984) 40:185-232). The only site containing melatoninla receptors in all photoperiodic species examined to date (Weaver, etal., Suprachiasmatic nucleus: the mind's clock. Klein, D. C., Moore, R.Y, and Reppert, S. M., eds. New York: Oxford University Press; (1991)pp. 289-308) is the pars tuberalis (PT), a portion of the pituitarygland. In contrast to other species, in humans melatonin la receptorsare not consistently present in the PT.

High-affinity melatonin-1a (Mel-1a ) receptors are located in discreteregions of the vertebrate central nervous system of several mammalianspecies, including humans. Binding studies using the ligand 2- ¹²⁵I!-iodomelatonin (¹²⁵ I-melatonin or ¹²⁵ I!MEL) have identifiedhigh-affinity melatonin la receptors (K_(d) <2×10⁻¹⁰ M) in sites such asthe suprachiasmatic nuclei (SCN), the site of a biological clock thatregulates numerous circadian rhythms (Reppert et al., Science (1988)242:78-81). To date, high-affinity melatonin receptors have not beenidentified in central nervous system tissues other than brain.

Receptor affinity is sensitive to guanine nucleotides and activation ofthe receptors consistently leads to the inhibition of adenylyl cyclasethrough a pertussis toxin-sensitive mechanism (Rivkees, S. A. et al.,P.N.A.S. USA (1989) 86:3883-3886; Carlson, L. L. et al., Endocrinology(1989) 125:2670-2676; Morgan, P. J. et al., Neuroendocrinology (1989)50:358-362; Morgan, P. J. et al., J. Neuroendocrinol. (1990) 2:773-776;Laitinen, J. T. and Saavedra, J. M., Endocrinology (1990)126:2110-2115). High-affinity melatonin receptors thus appear to belongto the superfamily of G protein-coupled receptors.

SUMMARY OF THE INVENTION

In general, the invention features substantially pure DNA (cDNA orgenomic DNA) encoding a high-affinity melatonin 1a receptor in brain andmelatonin 1b receptor in retina. The invention also featuressubstantially pure high-affinity melatonin 1a and 1b receptorpolypeptides. In preferred embodiments, the receptor includes an aminoacid sequence substantially identical to the amino acid sequence shownin FIG. 1 (SEQ ID NO:2); FIG. 2 (SEQ ID NO:4); FIG. 3 (SEQ ID NO:14);FIG. 5 (SEQ ID NO:12) or comprising the amino acid sequence of FIG. 4(SEQ ID NO:6) for melatonin-1a receptors.

The invention also features a new class of melatonin receptor designatedmelatonin-1b (Mel-1b ) distinguished by its tissue distribution andbinding characteristics. In preferred embodiments, the Mel-1b receptorincludes an amino acid sequence substantially identical to the aminoacid sequence shown in FIG. 6 (SEQ ID NO:16).

The invention includes a polypeptide having an amino acid sequence whichincludes a domain capable of binding melatonin and bringing about adecrease in intracellular cAMP concentration, and which is at least 80%identical to the amino acid sequence shown in FIGS. 1-6. The inventionalso features a substantially pure polypeptide which is a fragment oranalog of a high-affinity melatonin-1a or melatonin-1b receptor andwhich includes a domain capable of binding melatonin and bringing abouta decrease in intracellular cAMP concentration.

In various preferred embodiments, the receptor or receptor fragment isderived from a vertebrate animal, preferably, human, sheep, mouse, orXenopus laevis.

By "high-affinity melatonin receptor polypeptide" is meant all or partof a vertebrate cell surface protein which specifically binds melatoninand signals the appropriate melatonin-mediated cascade of biologicalevents (e.g., a decrease in intracellular cAMP) concentration. Thepolypeptide is characterized as having the ligand binding properties(including the agonist and antagonist binding properties) and tissuedistribution described herein.

By a "polypeptide" is meant any chain of amino acids, regardless oflength or post-translational modification (e.g., glycosylation).

By "substantially pure" is meant that the high-affinity melatoninreceptor polypeptide provided by the invention is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, high-affinity melatonin receptor polypeptide. Asubstantially pure high-affinity melatonin receptor polypeptide may beobtained, for example, by extraction from a natural source; byexpression of a recombinant nucleic acid encoding a high-affinitymelatonin receptor polypeptide, or by chemically synthesizing theprotein. Purity can be measured by any appropriate method, e.g., columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

By a "substantially identical" amino acid sequence is meant an aminoacid sequence which differs only by conservative amino acidsubstitutions, for example, substitution of one amino acid for anotherof the same class (e.g., valine for glycine, arginine for lysine, etc.)or by one or more non-conservative amino acid substitutions, deletions,or insertions located at positions of the amino acid sequence which donot destroy the biological activity of the receptor. Such equivalentreceptors can be isolated by extraction from the tissues or cells of anyanimal which naturally produce such a receptor or which can be inducedto do so, using the methods described below, or their equivalent; or canbe isolated by chemical synthesis; or can be isolated by standardtechniques of recombinant DNA technology, e.g., by isolation of cDNA orgenomic DNA encoding such a receptor.

By "derived from" is meant encoded by the genome of that organism andpresent on the surface of a subset of that organism's cells.

In another related aspect, the invention features isolated DNA whichencodes a high-affinity melatonin-1a or melatonin-1b receptor (orreceptor fragment or analog thereof) described above. Preferably, thepurified DNA is cDNA; is cDNA which encodes a Xenopus laevishigh-affinity melatonin receptor; is cDNA which encodes a sheephigh-affinity melatonin-1a receptor; and is cDNA which encodes a humanhigh-affinity melatonin-1a; and is cDNA which encodes a humanhigh-affinity melatonin-1b receptor.

By "isolated DNA" is meant a DNA that is not immediately contiguous with(i.e., covalently linked to) both of the coding sequences with which itis immediately contiguous (i.e., one at the 5' end and one at the 3'end) in the naturally-occurring genome of the organism from which theDNA of the invention is derived. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote; or which exists as a separate molecule (e.g., acDNA or a genomic or cDNA fragment produced by PCR or restrictionendonuclease digestion) independent of other sequences. It also includesa recombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In other related aspects, the invention features vectors which containsuch isolated DNA and which are preferably capable of directingexpression of the protein encoded by the DNA in a vector-containingcell; and cells containing such vectors (preferably eukaryotic cells,e.g., CHO cells (ATCC; Cat. No. CCL 61 or COS-7 cells (ATCC; Cat. No.CRL 1651). Preferably, such cells are stably transfected with suchisolated DNA.

By "transformed cell" is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of genetic engineering, a DNAmolecule encoding a high-affinity melatonin receptor (or a fragment oranalog, thereof). Such a DNA molecule is "positioned for expression"meaning that the DNA molecule is positioned adjacent to a DNA sequencewhich directs transcription and translation of the sequence (i.e.,facilitates the production of the high-affinity melatonin receptorprotein, or fragment or analog, thereof).

By "specifically binds", as used herein, is meant an agent, such asmelatonin, a melatonin analog or other chemical agent includingpolypeptides such as an antibody, which binds high-affinity melatoninreceptor, receptor polypeptide or a fragment or analog thereof, butwhich does not substantially bind other molecules in a sample, e.g., abiological sample, which naturally includes a high-affinity melatoninreceptor polypeptide. Preferably, the agent activates or inhibits thebiological activity in vivo of the protein to which it binds. By"biological activity" is meant the ability of the high-affinitymelatonin receptor to bind melatonin and signal the appropriate cascadeof biological events (as described herein).

In yet another aspect, the invention features a method of screeningcandidate compounds for their ability to act as an agonist of ahigh-affinity melatonin-1a or melatonin-1b receptor ligand. The methodinvolves:

a) contacting a candidate agonist compound with a recombinanthigh-affinity melatonin receptor (or melatonin-binding fragment oranalog);

b) measuring binding of the ligand to the receptor, the receptorpolypeptide or the receptor fragment or analog; and

c) identifying agonist compounds as those which bind the recombinantreceptor and trigger a decrease in intracellular cAMP concentration.

By an "agonist" is meant a molecule which mimics a particular activity,in this case, the ability of a high-affinity melatonin receptor ligandto bind a high-affinity melatonin receptor and to trigger the biologicalevents resulting from such an interaction (e.g., decreased intracellularcAMP concentration). An agonist may possess greater activity than thenaturally-occurring high-affinity melatonin receptor ligand.

In yet another aspect, the invention features a method of screening acandidate compound for its ability to antagonize interaction betweenmelatonin and a high-affinity melatonin receptor. The method involves:

a) contacting a candidate antagonist compound with a first compoundwhich includes a recombinant high-affinity melatonin receptor (ormelatonin-binding fragment or analog) on the one hand and with a secondcompound which includes melatonin on the other hand;

b) determining whether the first and second compounds bind; and

c) identifying antagonistic compounds as those which interfere with thebinding of the first compound to the second compound and which reducemelatonin-mediated decreases in intracellular cAMP concentration.

By an "antagonist" is meant a molecule which inhibits a particularactivity, in this case, the ability of melatonin to interact with ahigh-affinity melatonin receptor and to trigger the biological eventsresulting from such an interaction (e.g., decreased intracellular cAMPconcentration.)

In preferred embodiments of both screening methods, the recombinanthigh-affinity melatonin receptor is stably expressed by a mammalian cellwhich normally presents substantially no high-affinity melatoninreceptor on its surface (i.e., a cell which does not exhibit anysignificant melatonin-mediated decrease in intracellular cAMPconcentration); the mammalian cell is a CHO cell or a COS-7 cell; andthe candidate antagonist or candidate agonist is a melatonin analog orother chemical agent including a polypeptide such as an antibody.

The receptor proteins of the invention are likely involved in thecontrol of vertebrate circadian rhythm. Such proteins are thereforeuseful to develop therapeutics to treat such conditions as jet lag,facilitate reentrainment of some endogenous melatonin rhythms,synchronize the disturbed sleep-wake cycle of blind people, alleviatesleep disorders in shift workers, facilitate the emergence of a diurnalsleep-wake pattern in neonates, regulate ovarian cyclicity in humanfemales, control the initiation and timing of puberty in humans, andalter the mating cycle in seasonally breeding animals, such as sheep.Preferred therapeutics include 1) agonists, e.g., melatonin analogs orother compounds which mimic the action of melatonin upon interactionwith the high affinity melatonin receptor; and 2) antagonists, e.g.,melatonin analogs, antibodies, or other compounds, which block melatoninor high-affinity melatonin receptor function by interfering with themelatonin:receptor interaction.

A "transgenic animal" as used herein denotes an animmal (such as anon-human mammal) bearing in some or all of its nucleated cells one ormore genes derived from a different species (exogenous); if the cellsbearing the exogenous gene include cells of the animal's germline, thegene may be transmissible to the animal's offspring. As used herein,genes derived from a different species of animal are exogenous genes.Preferably the exogenous genes include nucleotide sequences which effectexpression of the gene in its endogenous tissue distribution.

Because the receptor component may now be produced by recombinanttechniques and because candidate agonists and antagonists may bescreened using transformed, cultured cells, the instant inventionprovides a simple and rapid approach to the identification of usefultherapeutics. Such an approach was previously difficult because of thelocalization of the receptor to a few discrete regions in the centralnervous system of most mammals. Isolation of the high-affinity melatoninreceptor gene (as cDNA) allows its expression in a cell type which doesnot normally bear high-affinity melatonin receptors on its surface,providing a system for assaying a melatonin:receptor interaction.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DETAILED DESCRIPTION

The drawings will first briefly be described.

Drawings

FIG. 1 is the complete nucleotide and amino acid sequences (SEQ ID NO:1and SEQ ID NO:2, respectively) of the Xenopus laevis high-affinitymelatonin receptor gene coding region cDNA. The deduced amino acidsequence of the receptor is provided below the nucleotide sequence(reading frame b) and contains 420 amino acids. The deduced amino acidsequence begins at nucleotides 32, 33, 34 (ATG=Met) and ends withnucleotides 1292, 1293, 1294 (TGA=stop).

FIG. 2 is the complete nucleotide and amino acid sequences (SEQ ID NO:3and SEQ ID NO:4, respectively) of the sheep high-affinity melatonin-1areceptor gene coding region which is a genetic fusion of genomic DNAfrom the 5' region and cDNA from the 3' region as described below. Thededuced amino acid sequence of the receptor is provided below thenucleotide sequence and contains (reading fame a) 366 amino acids. Thededuced amino acid sequence begins at nucleotides 49, 50, 51 (ATG=Met)and ends at nucleotides 1147, 1148, 1149 (TAA=stop).

FIG. 3 is the complete nucleotide and amino acid sequences (SEQ ID NO:13and SEQ ID NO:14, respectively) of the mouse high-affinity melatonin-1areceptor gene coding region. The deduced amino acid sequence of thereceptor is provided below the nucleotide sequence and contains (readingframe a) 353 amino acids. The deduced amino acid sequence begins atnucleotides 1-3 (ATG=Met) and ends at nucleotides 1060-1062 (TAA=stop).

FIG. 4 is the nucleotide and deduced amino acid sequences (SEQ ID NO:5and SEQ ID NO:6, respectively) of a fragment of the human high-affinitymelatonin receptor gene coding region genomic DNA. The coding sequencecorresponds to the region downstream (3') of the first intron. From thesequenced portion of the receptor DNA, the deduced amino acid sequenceis provided below the nucleotide sequence (reading frame a) and contains288 amino acids. The coding region of the partial sequence begins atnucleotides 1, 2, 3 (GGA=Gly) and ends at nucleotides 865, 866, 867(TAA=stop).

FIG. 5 is the complete nucleotide and amino acid sequences (SEQ ID NO:11and SEQ ID NO:12, respectively) of the human high-affinity melatoninreceptor cDNA. The deduced amino acid sequence of the receptor isprovided below the nucleotide sequence (reading frame c) beginning atnucleotides 33-35 (ATG=Met) and contains 350 amino acids ending atnucleotides 1083-1085 (TAA=stop).

FIG. 6 is the complete nucleotide and amino acid sequences (SEQ ID NO:15and SEQ ID NO:16, respectively) of the human high-affinity melatonin-1breceptor cDNA. The deduced amino acid sequence of the receptor isprovided below the nucleotide sequence (reading frame a) beginning atnucleotides 13-15 (ATG=Met), ending at nucleotides 1096-1098 (TAA=stop)and contains amino 362 acids.

FIG. 7 shows the alignment of the deduced amino acid sequences (SEQ IDNO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively) and the hydrophobicregions (boxes I-VII) of the entire Xenopus and sheep, and partial humanhigh-affinity melatonin receptors.

FIG. 8 shows the alignment of the deduced amino acid sequences (SEQ IDNO:2, SEQ ID NO:4, and SEQ ID NO:12, respectively) and the hydrophobicregions (presumed transmembrane domains I-VII highlighted by solid bars)of the entire Xenopus, sheep, and human high affinity melatoninreceptors. To indicate homology, gaps (represented by dots) have beenintroduced into the three sequences.

FIG. 9 is the proposed structure of the Xenopus high-affinity melatoninreceptor in the cell membrane. The deduced amino acid sequence (SEQ IDNO:2) is depicted. Y, potential N-linked glycosylation site. Solidcircles represent consensus sites for protein kinase C phosphorylation.

FIGS. 10a and 10b show ¹²⁵ I-melatonin binding assay results from COS-7cells containing Xenopus melatonin receptor cDNA. FIG. 11a shows asaturation curve. Nonspecific binding was determined using 10 μMmelatonin. FIG. 11b shows a single representative Scatchard plot of thesaturation data for determining the relative ¹²⁵ I-melatonin bindingconstants for the transfected high-affinity melatonin receptor gene fromXenopus.

FIG. 11 shows competition by various ligands for ¹²⁵ I-melatonin bindingin COS-7 cells transfected with the melatonin receptor cDNA fromXenopus. Cells were incubated with 100 pM ¹²⁵ I-melatonin and variousconcentrations of 2-iodomelatonin (I-MEL), melatonin (MEL),6-chloromelatonin (6Cl -MEL), 6-hydroxymelatonin (60H-MEL),N-acetyl-5-hydroxytryptamine (NAS), or 5-hydroxytryptamine (5HT).Nonspecific binding was determined in the presence of 10 μM melatonin.K_(i) values are: I-MEL, 1.1×10⁻¹⁰ M; MEL, 1.3×10⁻⁹ M; 6Cl-MEL, 3.0×10⁻⁹M; 60H-MEL, 2.0×10⁻⁸ M; NAS, 2.0×10⁻⁶ M; 5HT, >1.0×10⁻⁴ M. The data arerepresentative of three experiments.

FIG. 12 shows melatonin inhibition of forskolin-stimulated cAMPaccumulation in CHO cells stably transfected with the melatonin receptorcDNA from Xenopus. The 100% value is the mean cAMP value induced with 10μM forskolin. The data are representative of three experiments.

FIG. 13 is a Northern blot of melatonin receptor transcripts in Xenopusderived melanophores. Locations of RNA size markers (Life Technologies,Bethesda, Md.) are indicated. The blot was exposed to X-ray filmovernight.

FIG. 14 shows ¹²⁵ I-melatonin binding assay results from COS-7 cellscontaining sheep melatonin receptor cDNA. FIG. 14a shows a saturationcurve. Nonspecific binding was determined using 10 μM melatonin. FIG.14a (inset) shows a Scatchard plot of the saturation data fordetermining the relative ¹²⁵ I-melatonin binding constants for thetransfected high-affinity melatonin receptor gene from sheep. The K_(d)value for the sheep melatonin high-affinity receptor is 3.6×10⁻¹¹ M andthe B_(max) value is 104 fmol/mg protein. Nonspecific binding wasdetermined using 10 μM melatonin. Data shown are representative of threeexperiments. FIG. 14b is a plot of competition by various ligands for¹²⁵ I-Mel binding in COS-7 cells transfected with the sheep melatoninreceptor cDNA (SEQ ID NO:3). Cells were incubated with 100 pM ¹²⁵ I-Meland various concentrations of 2-iodomelatonin (I-Mel), melatonin (Mel),6-chloromelatonin (6Cl-Mel), 6-hydroxymelatonin (60H-Mel),N-acetyl-5-hydroxytryptamine (NAS), or 5-hydroxytryptamine (5-HT).Nonspecific binding was determined in the presence of 10 μM melatonin.K_(i) values for the sheep receptor are: I-Mel, 3.7×10⁻¹¹ M; Mel,2.4×10⁻¹⁰ M; 6Cl-Mel, 2.5×10⁻¹⁰ M; 60H-Mel, 3.0×10⁻⁹ M; NAS, 1.4×10⁻⁷ M;5HT, >1.0×10⁻⁴ M. Inhibition curves were generated by LIGAND (Munson, P.L. and Rodbard, D. Anal. Biochem. (1980) 107:220-239) using a one-sitemodel. The data shown are representative of at least three experiments.2-Iodomelatonin is available from Research Biochemicals Inc., Natick,Mass.; 6-chloromelatonin is available from Ely Lily, Indianapolis, Ind.;all other drugs used herein are available from Sigma, St. Louis, Mo.

FIG. 15 shows ¹²⁵ I-melatonin binding assay results from COS-7 cellscontaining the complete human melatonin 1a receptor cDNA (SEQ ID NO:11).FIG. 15a shows a saturation curve. FIG. 15a (inset) shows Scatchard plotof the saturation data for determining the relative ¹²⁵ I-melatoninbinding constants for the transfected high-affinity melatonin receptorgene from human. The K_(d) value for the human high-affinity melatonin1a receptor is 2.6×10⁻¹¹ M and the B_(max) value is 220 fmol/mg protein.Nonspecific binding was determined using 10 μM melatonin. Data shown arerepresentative of three experiments. FIG. 15b is a plot of competitionby various ligands for ¹²⁵ I-Mel binding in COS-7 cells transfected withthe human melatonin receptor cDNA (SEQ ID NO:11). Cells were incubatedwith 100 pM ¹²⁵ I-Mel and various concentrations of 2-iodomelatonin(I-Mel), melatonin (Mel), 6-chloromelatonin (6Cl-Mel),6-hydroxymelatonin (60H-Mel), N-acetyl-5-hydroxytryptamine (NAS), or5-hydroxytryptamine (5-HT). Nonspecific binding was determined in thepresence of 10 μM melatonin. K_(i) values for the human receptor are:I-Mel, 1.8×10⁻¹¹ M; Mel, 2.3×10⁻¹⁰ M; 6Cl-Mel, 2.0×10⁻⁹ M; 60H-Mel,2.0×10⁻⁹ M; NAS, 1.7×10⁻⁷ M; 5HT, >1.0×10⁻⁴ M. Inhibition curves weregenerated by LIGAND (Munson and Rodbard (1980), supra) using a one-sitemodel. The data shown are representative of at least three experiments.

FIG. 16 is the results of studies showing that recombinant mammalianmelatonin receptor couples to G_(i). FIG. 16a shows melatonin inhibitionof forskolin-stimulated cAMP accumulation in NIH 3T3 cells stablytransfected with the sheep melatonin receptor cDNA (SEQ ID NO:3). The100% value is the mean cAMP value induced with 10 μM forskolin. The datashown are representative of four experiments. FIG. 16b shows thatpertussis toxin blocks the ability of melatonin to inhibitforskolin-stimulated cAMP accumulation in NIH 3T3 cells stablytransfected with the sheep melatonin receptor cDNA (SEQ ID NO:3). Cellswere preincubated with either vehicle or pertussis toxin for 18 hours(PTX: 100 ng/ml; pertussis toxin was purchased from List, Campbell,Calif.). C, Basal levels; F, 10 μM forskolin alone; FM, 10 μM forskolinplus 1 μM melatonin. Data are the mean plus standard deviation for 3plates for each treatment. The data shown are representative of threeexperiments.

FIG. 17 shows a coronal section through the base of the sheep brain andpituitary. FIG. 17a is a histographic staining of the tissue sectionshowing the pars tuberalis (PT) and the pars distalis (PD). FIG. 17b isa film autoradiographic image produced from a section to which ¹²⁵ I!MELbinding is observed in the PT. FIG. 17c is a film autoradiographic imageproduced from an in situ hybridization of a tissue section using a sheephigh-affinity melatonin receptor riboprobe derived from the clonedreceptor sequence. The hybridization pattern shows that mRNA whichhybridizes to the sheep high-affinity melatonin receptor riboprobeexhibits the same pattern of expression as the endogenous receptorprotein.

FIG. 18 is a diagram of the structure of the human Mel-1b receptorprotein. FIG. 18a is the predicted membrane topology of the human Mel-1breceptor protein. Y, Potential N-linked glycosylation site. Amino acidsthat are shaded are identical between human Mel-1b and the human Mel-1amelatonin receptors. FIG. 18b is a comparison of the deduced amino acidsequence of human Mel-1b and the human Mel-1a melatonin receptor(GenBank accesssion no. U14109) and the Xenopus melatonin receptor(U09561). To maximize homologies, gaps (dots) have been introduced intothe three sequences. The seven presumed transmembrane domains (I-VII)are overlined. Consensus sites for N-linked glycosylation areunderlined. The human melaton 1b receptor sequence has been deposited inGenBank under accession number U25341.

FIG. 19 is a plot of human Mel-1b receptor expression in COS-1 cellsassayed by ¹²⁵ I-Mel binding. ∘, total binding; , specific binding; ▴,nonspecific binding (determined in the presence of 10 μM melatonin).Inset: Scatchard plot of saturation data. The K_(d) value depicted is1.5×10⁻¹⁰ M. The B_(max) value is 2.62 pmol/mg membrane protein. Datashown are representative of five experiments.

FIG. 20 is a graphical representation of competition by various ligandsfor ¹²⁵ I-Mel binding in COS-1 cells transfected with either humanMel-1b or human Mel-1a melatonin receptor cDNA. Cells were incubatedwith 200 pM (Mel-1b receptor) or 100 pM ¹²⁵ I-Mel (Mel-1a receptor) andvarious concentrations of 2-iodomelatonin (I-Mel), melatonin (Mel),6-chloromelatonin (6Cl-Mel), or N-acetyl-5--hydroxytryptamine (NAS).Nonspecific binding was determined in the presence of 10 μM melatonin.The data shown are mean values of three to five experiments for eachdrug. K_(i) values are listed in Table 1.

FIG. 21 is a graphical representation of melatonin inhibition offorskolin-stimulated cAMP accumulation in NIH 3T3 cells stablytransfected with human Mel-1b receptor. The 100% value is the mean cAMPvalue induced with 10 μM forskolin. The data shown are mean values oftwo experiments.

FIG. 22 is a comparative RT-PCR analysis of Mel-1b and Mel-1a receptorgene expression in six human tissues. Brain refers to analysis of wholebrain. H3.3 is histone H3.3.

FIG. 23 is a diagram showing the chromosomal location of the Mel-1breceptor gene. The idiogram of human chromosome 11 illustrates thechromosomal content of the somatic cell hybrids used to localize theMel-1b melatonin receptor gene (MTNR1 B), to 11q21-22.

There now follows a description of the cloning and characterization ofthe high-affinity melatonin receptor cDNA from Xenopus laevis, thehigh-affinity melatonin 1a receptor from sheep, mouse, and human as wellas the high affinity melatonin 1b receptor from human useful in theinstant invention. Transformed cells containing and expressing the cDNAof the invention are also described. This example is provided for thepurpose of illustrating the invention, and should not be construed aslimiting.

Molecular Cloning of a High-Affinity Melatonin Receptor from Xenopuslaevis

Melatonin receptors are present in the dermal melanophores of amphibians(Bagnara, J. T. and Hadley, M. E., Am. Zoologist (1970) 10:201-216). Theaction of melatonin, mediated through the high-affinity melatoninreceptor coupled to G_(i) protein (Abe, K. et al., Endocrinology (1969)85:674-682; White, B. H. et al., J. Comp. Physiol. (1987) B 157:153-159)results in melanin aggregation in the dermal melanophores. mRNA fromXenopus dermal melanophores was used to clone the Xenopus high-affinitymelatonin receptor cDNA (Ebisawa, T. et al., PNAS USA (1994)91:6133-6137). Either primary cells or immortalized cells may be usedfor the purpose of mRNA isolation. Cloning of the Xenopus high-affinitymelatonin receptor cDNA was accomplished as a useful initial step towardcloning of the high-affinity melatonin receptors of higher eukaryotes.

The immortalized cell line used for mRNA isolation was found to expressa high level of ¹²⁵ I-melatonin binding (≧100 fmol/mg total cell proteinusing 50 pM ¹²⁵ I-melatonin). The cells were cultured by the method ofDaniolos et al. (Pigment Cell Res. (1990) 3:38-43). Using standardtechniques, total cellular RNA was isolated from melanophores byextraction with guanidinium thiocyanate followed by centrifugalseparation in a cesium chloride density gradient (Sambrook et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbour Lab. Press,Plainview, N.Y.), (1989) 2nd Ed.). Removal of melanosomes prior toseparation on the cesium chloride density gradient was performed asdescribed by Karne et al. (J. Biol. Chem. (1993) 268:19126-19133).Poly(A)⁺ RNA was isolated using established methods as described inRivkees et al. (P.N.A.S. USA (1989) 84:3916-3920).

The poly(A)⁺ RNA from Xenopus dermal melanophores was used as a templatefor the construction of a random primed cDNA library (cDNA SynthesisKit, Pharmacia Biotech Inc., Piscataway, N.J.). Cohesive ends wereproduced on the double stranded cDNA by ligating with BstX1 and EcoR1adaptors (InVitrogen, San Diego, Calif.). The cDNA was size-fractionatedon an agarose gel, and cDNA having a length equal to or greater than 2kilobase pairs (kb) was recovered by electroelution. The size-selectedcDNA was ligated into the expression vector pcDNAI (InVitrogen, SanDiego, Calif.) and introduced into E. coli strain MC1061/P3 byelectroporation.

A total of 4×10⁵ recombinants were obtained from 5 μg of poly(A)⁺ RNAand divided into 54 pools, each containing approximately 7400 clones.Plasmid DNA was prepared from each pool by the alkaline lysis method andtransfected into COS-7 cells by the DEAE-dextran method (Cullen, B. R.,Methods Enzymol. (1987) 152:684-704). COS-7 cells were grown asmonolayers in Dulbecco's modified Eagle's medium supplemented with 10%fetal calf serum, penicillin (50 U/ml), and streptomycin (50 μg/ml), in5% CO₂ at 37° C. Three days after transfection, cells were incubatedwith 90 pM ¹²⁵ I-melatonin Tris-HCl pH 7.4, containing 100 mM NaCl, 5 mMKCl, 2 mM CaCl₂, and 5% Nu-Serum I (Collaborative Biomedical Products,Bedford, Mass.), for 2 hr at room temperature. Cells were washed, airdried, and exposed to X-ray film for 14 days. A pool of clones whichshowed positive signals was subdivided, and the transfection procedurewas repeated. This subdividing process was continued until a singleclone was identified that conferred specific ¹²⁵ I-melatonin binding toCOS-7 cells.

This clone, which contained a 2.2 kb cDNA, insert was isolated and bothstrands of the coding region were sequenced (SEQ ID NO:1). Nucleotidesequences were analyzed by the dideoxynucleotide chain terminationmethod of Sanger, F. et al. (P.N.A.S. USA (1977) 74:5463-5467) usingSequenase® (United States Biochemical, Cleveland, Ohio). The sequencingtemplate was double-stranded plasmid DNA. Sequencing primers weresynthetic oligonucleotides that were either vector specific or derivedfrom sequence information.

The isolated Xenopus cDNA encodes a protein of 420 amino acids (FIG. 1)(SEQ ID NO:2) with an estimated molecular mass of 47,424. The flankingDNA sequence of the first two methionine codons in this reading frameboth displayed a Kozak consensus sequence for the initiation oftranslation (Kozak, M., Nucleic Acids Res. (1987) 15:8125-8148).Hydropathy analysis (Kyte, J. and Doolittle, R. F., J. Mol. Biol. (1982)157:195-232) of the predicted amino acid sequence revealed the presenceof seven hydrophobic domains (see FIGS. 4 and 5) which likely representthe transmembrane regions of a G protein-coupled receptor. The aminoterminus contains a consensus site for N-linked glycosylation, a featuretypical of most G protein-coupled receptors (Pearson, W. R., MethodsEnzymol. (1990) 183:63-98). The melatonin receptor protein is notsimilar in identity to any one particular group of G protein-coupledreceptors, but is similar to a wide range of receptors; the highestamino acid sequence identity scores were approximately 25% for both themu opioid and type 2 somatostatin receptors. Using a G protein-coupledreceptor database (Kornfeld, R. and Kornfeld, D., Ann. Rev. Biochem.(1985) 54:631-664), the melatonin receptor appears to form a group thatis distinct from other known biogenic amine and peptide receptors. Nosequence homology was identified between the melatonin receptor and themetabotropic glutamate or parathyroid hormone/calcitonin/secretinreceptor gene families (Masu et al., Nature (1991) 349:760-765; Juppner,et al., Science (1991) 254:1024-1026; Lin et al., Science (1991)254:1022-1024).

The melatonin receptor has some general structural features in commonwith amine and peptide receptors. For example, it contains a singlecysteine residue in each of the first two extracellular loops that,based on mutagenesis studies of opsin and amine receptors (Dixon et al.,EMBO J. (1987) 6:3269-3275; Karnic et al., P.N.A.S. USA (1988)85:8459-8463), are believed to form a disulfide bridge which stabilizesreceptor structure. Furthermore, proline residues are present intransmembrane domains IV, V and VI (FIG. 7 and FIG. 8) which have beensuggested to introduce kinks in the alpha-helices that may be importantin forming the ligand binding pocket (Findlay, J. and Eliopoulos, E.,Trends Pharmacol. Sci. (1990) 11:492-499; Hibert, M. F. et al., Mol.Pharmacol. (1991) 40:8-15). The proline in the NPXXY (SEQ ID NO:7) motifthat is found in transmembrane domain 7 of virtually all other Gprotein-coupled receptors is replaced by an alanine in the melatoninreceptor. The carboxyl tail of the melatonin receptor is 119 amino acidresidues long and contains several consensus sites for protein kinase Cphosphorylation which may be involved in receptor regulation (Sibley etal., Cell (1987) 48:913-922).

Binding Studies of the Recombinant Xenopus High-Affinity MelatoninReceptor.

To establish the binding characteristics of the encoded Xenopus receptor(SEQ ID NO:2), the cDNA in pcDNAI was transiently expressed in COS-7cells. Three days after transfection, medium was removed, the culturedishes were washed with PBS, and the cells were harvested. The cellswere then pelleted (2500 rpm; 10 min, 4° C.) and stored at -80° C. Wholecell binding studies were performed by thawing the cells andresuspending them in binding buffer (50 mM Tris-HCl, pH 7.4, with 5 mMMgCl₂) at a concentration of 456 μg protein/ml. The cell suspension wasincubated with ¹²⁵ I-melatonin (90 pm) in a total reaction volume of 0.2ml binding buffer in the presence or absence of a melatonin agonist orantagonist; the suspension was incubated in a shaker bath for 1.5 hr at25° C. Protein determinations were performed using the Pierce BCAProtein Assay (The Pierce Chemical Co., Rockford, Ill.). Binding datawere analyzed by computer using the LIGAND Program of Munson and Rodbard((1980) supra). The results are shown in FIGS. 8 and 9.

To further establish the binding characteristics of the encoded Xenopusreceptor (SEQ ID NO:2), the cDNA in pcDNAI was transiently expressed inCOS-7 cells. Three days after transfection, saturation studies wereperformed using increasing concentrations of ¹²⁵ I-melatonin (5 to 1280pM) (FIG. 10a). Scatchard analysis (FIG. 10b) revealed that transfectedCOS-7 cells bound ¹²⁵ I-melatonin with high affinity (K_(d) =63±3×10⁻¹²; n=3 experiments). The B_(max) value using the whole cell binding assaywas 67±7 fmol/mg of protein. No specific binding of ¹²⁵ I-melatonin wasfound in mock-transfected COS-7 cells.

The pharmacological characteristics of specific ¹²⁵ I-melatonin bindingin acutely transfected COS-7 cells was next examined (FIG. 11). Theorder of inhibition of specific ¹²⁵ I-melatonin binding of Xenopusrecombinant melatonin receptor by six ligands was characteristic of ahigh-affinity melatonin receptor (Dubocovich, M. L. and Takahasi, J.(1987) supra; Rivkees et al. (1989) supra), with relative bindingaffinities having the order:2-iodomelatonin>melatonin>6-chloromelatonin>6-hydroxymelatonin>n-acetyl-5-hydroxytryptamine>5-hydroxytryptamine.Thus, the isolated Xenopus laevis cDNA of the instant invention encodesa protein with the affinity and pharmacological properties expected of ahigh-affinity melatonin receptor.

The endogenous high-affinity melatonin receptor in Xenopus dermalmelanophores is coupled to inhibition of adenylyl cyclase (Abe, K. etal. (1969) supra; White, B. H. et al. (1987) supra). To determinewhether the receptor encoded by the recombinant cDNA (SEQ ID NO:1) ofXenopus was coupled to the adenylyl cyclase regulatory system, a clonalline of CHO (ATCC; Cat. No. CCL 61 cells) was stably transfected withthe recombinant receptor cDNA and the melatonin-induced inhibition offorskolin-stimulated cAMP accumulation was determined.

Transformed CHO cells were plated on 35 mm culture dishes. After 48hours, the cells were washed twice with Ham's F-12 (Life Technologies,Bethesda, Md.). Cells were then incubated in the presence or absence ofmelatonin analogs (diluted in F-12) for 10 min at 37° C. Followingtreatment, the medium was aspirated and 1 ml of 50 mM acetic acid wasadded to the culture dish. The cells were collected, transferred to anEppendorf tube, boiled for 5 min, and centrifuged (13,750 rpm for 15min). The supernatant was collected and assayed for cAMP. Alldeterminations were performed in triplicate. Cyclic AMP levels weredetermined in duplicate by radioimmunoassay (New England Nuclear,Boston, Mass.).

Induction of cAMP concentration increase by 10 μM forskolin wasinhibited by melatonin in a dose dependent manner (FIG. 12); the maximalinhibition of the mean forskolin-stimulated cAMP concentration was 68%at 1×10⁻⁸ M melatonin. An IC₅₀ value of approximately 8×10⁻¹⁰ M wasdetermined by manual curve fitting of the data in FIG. 12. This valuewas very similar to the computer-generated K_(i) value (1.3×10⁻⁹ M)determined for melatonin inhibition of specific ¹²⁵ I-melatonin bindingshown in FIG. 11. Melatonin, alone, (1×10⁻⁶ M) was found not to alterbasal cAMP levels in stably transfected CHO cells. Further, melatonin(1×10⁻⁶ M) did not inhibit the forskolin-stimulated increase in cAMPlevels in CHO cells stably transfected with vector lacking the XenopuscDNA. Thus, the recombinant melatonin receptor is negatively coupled tothe cAMP regulatory system.

Expression of Xenopus Melatonin Receptor Transcripts

Northern blot analysis (see below) of Xenopus dermal melanophoresrevealed at least 3 hybridizing transcripts between 2.4 and 4.4 kb underconditions of high stringency (see below) (FIG. 13). The presence ofmultiple hybridizing bands may represent posttranscriptionalmodifications of the same gene, or the presence of transcripts fromdifferent, but structurally similar genes.

Northern analysis was performed using standard techniques (see, e.g.,Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, (1989)). Poly(A)⁺ RNA was subjected to electrophoresisthrough a 1% agarose-formaldehyde gel, blotted onto GeneScreen (NewEngland Nuclear, Boston, Mass.), and hybridized with a fragment of thecoding region of the receptor cDNA labeled with α--³² P!dCTP (2000Ci/mmol) by the method of random priming (Promega, Madison, Wis.).Hybridizing conditions were 50% formamide, 1M sodium chloride, 1% SDS,10% dextran sulfate, and 100 μg/ml denatured salmon sperm DNA, at 42° C.overnight. The final washing of the blot was in 0.2X SSC/0.1% SDS at 65°C. for 40 min. Blots were exposed at -80° C. to X-ray film with anintensifying screen.

Isolation of Sheer High-Affinity Melatonin Receptor

To clone the high-affinity melatonin receptor from sheep using standardmethods, fully degenerate primers were designed based on, for example,the peptide sequences 5' AIAINRY (SEQ ID NO:8) (residues 125-131) and 3'FAVCWAPL (SEQ ID NO:9) (residues 252-259) of the Xenopus sequence (SEQID NO:1) (FIG. 1). Using these populations of degenerate primers, RT PCRof sheep pars tuberalis mRNA amplified an approximately 400 bp cDNAfragment that was 65% identical at the amino acid level with thecorresponding region of the Xenopus melatonin receptor.

To isolate a longer cDNA sequence, this fragment was labeled (e.g., with³² P!dCTP by random priming) producing a probe, and hybridization (underhigh stringency conditions) was carried out on a sheep pars tuberaliscDNA library constructed in the ZAP II vector (Stratagene, La Jolla,Calif.) using standard hybridization techniques (see e.g., Ausubel etal., Current Protocols in Molecular Biology, supra). From 1×10⁶recombinants screened, two hybridizing clones were isolated and plaquepurified using standard techniques. Both clones contained the entire 3'coding region, downstream from the predicted site of the thirdtransmembrane domain. One clone extended 5' into the amino terminusregion, upstream from the first transmembrane domain, but did notcontain the entire 5' end of the coding region. A 160 bp fragment of the5' end of this cDNA clone was labelled (e.g., radiolabelled) by standardtechniques (see e.g., Ausubel et al., supra) and used to probe (e.g., bythe standard techniques described, supra) a sheep genomic library (inEMBL-3, catalog number UL 1001d, Clontech, Palo Alto, Calif.). One clonewas isolated and found to contain the remaining 5' sequence of thecoding region using standard sequencing techniques. A 150 bp fragment ofthis genomic clone, containing a methionine with a consensus sequencefor the initiation of translation was isolated and ligated usingstandard techniques (see e.g., Sambrook (1989), supra) into a vector(e.g., pcDNAI, InVitrogen, San Diego, Calif.) in frame with thecorresponding downstream coding region of the cDNA. The ligatedconstruct encodes a protein of 366 amino acids (SEQ ID NO:4) which binds¹²⁵ I!MEL with high affinity.

Binding Studies of the Recombinant Sheep High-Affinity MelatoninReceptor

The sheep high-affinity melatonin 1a receptor (SEQ ID NO:3) DNA clonedinto pcDNAI was transiently expressed in COS-7 cells. For ligand bindingstudies, the sheep receptor cDNA (SEQ ID NO:3) in pcDNAI was introducedinto COS-7 cells using the DEAE-dextran method (Cullen, B. R. MethodsEnzymol. (1987) 152:684-704). Approximately two to three days aftertransfection, cell culture medium was removed, the cultures dishes werewashed with PBS, and the cells were harvested. The cells were thenpelleted (2500 rpm; 10 min, 4° C.) and stored at -80° C. Whole cellbinding studies were performed by thawing the cells and resuspendingthem in binding buffer (50 mM Tris-HCl, pH 7.4, with 5 mM MgCl₂) at aconcentration of 200-500 μg protein/ml. The cell suspension wasincubated with ¹²⁵ I-Mel with or without drugs in a total reactionvolume of 0.2 ml binding buffer; the suspension as incubated in a shakerbath for 1.5 hr at 25° C. All determinations were done in eitherduplicate or triplicate. Protein measurements were performed using thePierce BCA Protein Assay. Binding data were analyzed by computer usingthe LIGAND Program of Munson and Rodbard (1980).

Scatchard analysis (performed as described above for the Xenopus clone)revealed that COS-7 cells transfected with the sheep Mel-1a receptorclone bound ¹²⁵ I-melatonin with high affinity (K_(d) =3.6±0.1×10⁻¹¹ M;mean ±SE, n=3 experiments). The B_(max) value for the sheep receptorclone using the whole cell binding assay was greater than 112±5 fmol/mgof protein (FIG. 14a). No specific binding of ¹²⁵ I-melatonin was foundin mock-transfected COS-7 cells.

The sheep Mel-1a receptor pharmacologic profile of relative bindingaffinities of melatonin derivatives was shown to be similar to Xenopususing the same assay techniques as described for Xenopus. Competitivebinding of six ligands to sheep melatonin receptor expressed by acutelytransfected COS-7 cells showed that the rank order of inhibition ofspecific ¹²⁵ I-Mel binding by the six ligands was2-iodomelatonin>melatonin=6-chloromelatonin>6-hydroxymelatonin>N-acetyl-5-hydroxytryptamine>5-hydroxytryptamine(FIG. 14b).

The receptor encoded by the recombinant sheep melatonin 1a receptor wastested to determine whether it is coupled to inhibitory G protein(G_(i)), as has been shown with the endogenous receptor of severalmammals, including sheep (Carlson, et al., (1989) supra; Morgan et al.,(1990) supra). Clonal NIH 3T3 cells stably transfected with the sheepreceptor CDNA (SEQ ID NO:3) subcloned into pcDNAI NEO (Invitrogen, SanDiego, Calif.) and exhibiting high levels of melatonin receptor binding(>10 fmol/60 mm dish of cells using 100 pM ¹²⁵ I-Mel) were used.Transformed NIH 3T3 cells were plated on 35 mm dishes. After forty-eighthours, the cells were washed twice with DMEM, and then incubated with orwithout drugs (diluted in DMEM) for 10 min at 37° C. At the end oftreatment, the medium was aspirated and 1 ml of 50 mM acetic acid wasadded. The cells were collected, transferred to an Eppendorf tube,boiled for 5 min, and centrifuged (13,750 rpm for 15 min). Thesupernatant was collected and assayed for cAMP. All determinations weredone in triplicate. Cyclic AMP levels were determined in duplicate byradioimmunoassay by standard techniques.

Although melatonin did not alter basal cAMP levels in the stablytransfected lines, it did cause a dose-dependent inhibition of the cAMPincrease induced by 10 μM forskolin (FIG. 16a). The estimated IC₅₀ valuefor melatonin was 1×10⁻¹⁰ M, comparable to the K_(i) value for melatonininhibition of specific ¹²⁵ I-Mel binding (2.4×10⁻¹⁰ M; see FIG. 14b).Importantly, melatonin (1 μM) did not inhibit forskolin-stimulated cAMPaccumulation in NIH 3T3 cells stably transfected with the vector (pcDNAINEO) lacking the sheep Mel-1a receptor cDNA.

Pertussis toxin pretreatment (PTX; 100 ng/ml) of receptor-transfectedNIH 3T3 cells for 18 hours completely abolished the ability of 1 μMmelatonin to inhibit the forskolin-stimulated increase in cAMP (FIG.16b). Thus, like the endogenous high-affinity melatonin receptor ofvertebrates (Carlson et al., (1989) supra; Morgan et al., (1990) supra;White et al., (1987) supra), the recombinant sheep Mel-1a receptorinhibits adenylyl cyclase through a pertussis-toxin sensitive mechanism.

Northern blot analysis of sheep PT revealed a major hybridizingtranscript of greater than 9.5 kb and a minor transcript at 4.2 kb. Nohybridizing signals were found in pars distalis (data not shown). Usingantisense cRNA probes prepared using sheep melatonin 1a receptor cDNA,in situ hybridization of endogenous mRNA revealed a strong hybridizationsignal that was visible in film autoradiographs of the sheep PT (FIG.17); no signal was detected in pars distalis. The mRNA distribution inPT was identical to that found for the receptor protein using ¹²⁵ I-Melin vitro autoradiography. The SCN region of sheep was not examined formelatonin receptor mRNA because high-affinity melatonin receptors havenot been identified in sheep SCN using ¹²⁵ I-Mel in in vitroautoradiography (Bittman, E. L. and Weaver, D. R., Biol. Reprod. (1990)43:986-993).

Brain tissue of Siberian hamster and rat were examined to illustrate thedistribution of melatonin receptor in brain of other species in whichmelatonin is known to have affects on reproductive and circadian rhythms(Bartness, T. J. et al., J. Pineal Res. (1993) 15:161-190; Margraf, R.R. and Lynch, G. R., Am. J. Physiol. (1993) 264:R615-R621; and Cassone,V. M., Trends Neurosci. (1990) 13:457-464). The major sites of specific¹²⁵ I-Mel binding and receptor transcript hybridization in Siberianhamster brain are the PT, SCN and paraventricular nucleus of thethalamus as examined in adjacent sections by in vitro autoradiography(data not shown; see also Weaver, D. R. et al., J. Neurosci. (1989)9:2582-2588). Thus, in this species, the distribution of melatonin 1areceptor mRNA and protein are identical and restricted to just a fewsites in brain. The PT and SCN regions exhibited receptor transcripthybridization and ¹²⁵ I-Mel binding in adult and developing rats (datanot shown). The distribution of melatonin 1a receptor mRNA wascoincident to that of ¹²⁵ I-Mel binding throughout the SCN in both ratand hamster.

In all non-human mammals we have examined, including the sheep (FIG.15), Siberian hamster, Syrian hamster, and rat, in situ hybridizationstudies have readily detected mRNA for the high-affinity melatonin 1areceptor in PT. The PT currently appears to be an important site throughwhich melatonin mediates photoperiodic effects on reproductive function.The PT is the only site containing melatonin 1a receptors (as detectedwith ¹²⁵ I-Mel in vitro autoradiography) in all seasonally breedingmammals examined to date (Weaver et al., (1991) supra). The mechanismsby which the PT processes the daily melatonin signal and communicatesthat information to influence hypothalamic neurosecretion are unknown.High-affinity melatonin receptors have not been consistently detected inthe human PT by ¹²⁵ I-Mel in vitro autoradiography, suggesting thatneuroendocrine responses to melatonin in humans may occur throughfundamentally different mechanisms than those that underlie theregulation of reproduction in seasonally breeding species (Weaver, D. R.et al., J. Clin. Endocrinol. Metab. (1993) 76:295-301.

Isolation of the Mouse High-Affinity Melatonin Receptor

Degenerate primers were designed using regions conserved amoung othermammalian Mel-1a receptor cDNAs such as those from sheep (see FIG. 2).Polymerase chain reaction (PCR) of mouse genomic DNA yielded a 466 bpfragment that was 94% identical at the amino acid level to the rat andDjungarian hamster Mel-1a receptor cDNAs. In situ hybridization of adultC57BL/6J mouse brain using the PCR-generated fragment produced ahybridization pattern consistent with that expected for the Mel-1amelatonin receptor. Hybridization signal was most intense in thehypophyseal pars tuberalis. Southern blot analysis of genomic DNAindicated a single-copy gene. RNA was isolated from a murine cell line(RT2-2) which expresses the Mel-1a receptor. Northern analysis ofpoly(A)⁺ RNA indicated a transcript length of approximately 1.9 kb.RT-PCR was used to generate the full length coding region (1059 bp) ofthe receptor, which showed 84% amino acid identity to the human Mel-1areceptor. RNase protection analysis, 5' and 3' RACE cloning, andscreening of a BALB/c mouse EMBL3 SP6/T7 genomic library revealed thatthe receptor gene consists of 2 exons divided by a large (>8 kb) intron.The 3' untranslated region is 444 bp long, and includes thepolyadenylation signal AUUAAA. RNase protection assays suggest that amajor transcription start site is located approximately 100 bp upstreamof the initiation codon. The nucleotide sequence and deduced amino acidsequence of the mouse Mel-1a receptor are shown in FIG. 3.

The recombinant mouse Mel-1a receptor expressed on COS-7 cells boundmelatonin with high affinity comparable to the binding affinity of sheepand human Mel-1a receptors.

Isolation of a Fragment of the Human High-Affinity Mel-1a Receptor

To clone the human high-affinity melatonin receptor, the degenerateprimers based on the peptide sequences 5' AIAINRY (SEQ ID NO:8)(residues 125-131 of the Xenopus deduced amino acid sequence (SEQ IDNO:2)) and 3' FAVCWAPL (SEQ ID NO:9) (residues 252-259 of the Xenopusdeduced amino acid sequence (SEQ ID NO:2)) were used as described above.Human genomic DNA was amplified by standard PCR techniques using thedegenerate primers and an approximately 400 bp fragment was isolated andsequenced by standard techniques. The deduced amino acid sequence of the400 bp fragment was 65% identical at the amino acid level with thecorresponding portion of the Xenopus high-affinity melatonin receptor.The 400 bp fragment was labelled (e.g., by random primer labelling; seee.g., Ausubel, supra) and used to screen a human genomic library (invector EMBL-3, Clontech, Palo Alto, Calif., catalog number HL1067J)under high stringency conditions using standard hybridization techniques(see, e.g., Ausubel, supra). Several positively hybridizing clones wereidentified from 1×10⁶ recombinant clones screened. The clones wereplaque purified by standard techniques, digested with appropriaterestriction enzymes and subcloned in to a convenient vector forsequencing (e.g., pBluescript®, Stratagene, La Jolla, Calif.). The humaninsert DNA (SEQ ID NO:5) of one clone was sequenced using standardtechniques. Using the sheep (SEQ ID NO:3) and Xenopus (SEQ ID NO:1)nucleotide and deduced amino acid sequences (SEQ ID NO:4 and SEQ IDNO:2, respectively) for comparison (see FIG. 7 and FIG. 8), the humaninsert DNA was found to contain a portion of the coding region from the"GNXFVV (SEQ ID NO:10) motif" just downstream from the firsttransmembrane domain (see FIGS. 7 and 8) and extends through the 3' endof the coding region. The human DNA of the sequenced clone isapproximately 82% identical to the sheep nucleotide sequence (SEQ INNO:5) of the corresponding region. The sheep and human deduced aminoacid sequences (SEQ IN NO:4 and SEQ IN NO:6, respectively) areapproximately 80% identical in the corresponding regions. Thus the humanDNA fragment (SEQ IN NO:5) isolated by the above techniques encodes aprotein with strong identity to the corresponding portion ofhigh-affinity melatonin receptor in another mammal, sheep.

The human genomic DNA contains an intron (>2.0 kb in length) upstream ofthe "GNXFVV motif" (SEQ IN NO:10). To obtain the 5' portion of thecoding region of the human receptor, the 160 bp fragment of the codingregion of the sheep receptor immediately upstream from this GNXFVV motifwas used to reprobe the human genomic library at low stringency (forexemplary low stringency hybridization conditions see e.g., Ausubel etal. (1989), supra). One positively hybridizing clone was isolated andfound by standard sequence analysis to contain the 5' end of the codingregion. RT-PCR (see e.g., Reppert, et al., Mol. Endocrinol. (1991)5:1037-1048) of mRNA from human hypothalamus using specific primersdirected at the 5' and 3' ends of the putative coding region amplifiedthe expected CDNA, containing the coding region of the human melatoninreceptor. The cDNA was subcloned into pcDNAI for sequence analysis andtransient expression of the receptor polypeptide.

The sequencing results show that cDNAs cloned in the instant inventionencode a high-affinity melatonin receptor from Xenopus sheep, and human.Overall, the coding regions of the sheep receptor and complete humanreceptor are about 60% identical with that of the Xenopus melatoninreceptor. Within the transmembrane domains, the identity is 77%. Themost dissimilar regions between the mammalian and frog receptors was inthe amino and carboxyl terminal regions. The amino terminus of themammalian receptors contains two consensus sites for N-linkedglycosylation, compared to one site in the frog receptor. Furthermore,the carboxyl tail of the sheep and human receptors is 65 amino acidresidues shorter than the Xenopus receptor tail. The complete humanhigh-affinity melatonin receptor DNA shows strong identity(approximately 82% at the nucleotide level and approximately 80% at theamino acid level) to the sheep high-affinity melatonin receptor with 87%amino acid identity when comparison is limited to the transmembranedomains. This high structural homology suggests that the human and sheepclones are species homologs of the same receptor.

Binding Studies of the Recombinant Human High-Affinity Mel-1a Receptor

The complete human high-affinity melatonin 1a receptor (SEQ ID NO:11)DNA cloned into pcDNAI was transiently expressed in COS-7 cells andbinding studies were performed as described for the sheep receptor,supra. Scatchard analysis (performed as described above for the Xenopusand sheep clones) revealed that COS-7 cells transfected with thecomplete human receptor clone (containing DNA of SEQ ID NO:11) bound ¹²⁵I-melatonin with high affinity (K_(d) =2.6 and 2.3×10⁻¹¹ M; n=2experiments). The B_(max) value using the whole cell binding assay was210 and 220 fmol/mg protein for the human receptor in two experiments(FIG. 15). No specific binding of ¹²⁵ I-melatonin was found inmock-transfected COS-7 cells. For the human clone, the rank order ofinhibition was identical to that for sheep, except that6-chloromelatonin was 10-fold less potent in inhibiting specific ¹²⁵I-Mel binding (k_(i) values listed in legend of FIG. 13b). Thus, therecombinant sheep and human receptors bind ¹²⁵ I-Mel with high affinityand exhibit the appropriate pharmacological characteristics of ahigh-affinity melatonin receptor (Dubocovich and Takahashi, (1987)supra; Morgan et al., (1989) J. Endocrinol. 1:1-4; Rivkees et al., PNASUSA (1989) 86:3883-3886; Vanecek, J., J. Neurochem. (1988)51:1436-1440).

Isolation of a Human High-Affinity Mel-1b Receptor.

To clone melatonin receptor subtypes, PCR was used to amplify humangenomic DNA with degenerate oligonucleotide primers based on conservedamino acid residues in the third and sixth transmembrane domains of theXenopus melatonin receptor and mammalian Mel-1a melatonin receptors.

For PCR with degenerate primers, genomic DNA was subjected to 30 cyclesof amplification with 200 nM (final concentration) each of twooligonucleotide primers. Each reaction cycle consisted of incubations at94° C. for 45 sec, 45° C. for 2 min and 72° C. for 2 min, with AmpiTaqDNA polymerase (Perkin-Elmer Cetus). The amplified DNA was separated onan agarose gel. DNA bands were subcloned into pCRTMII using a TA CloningKit (Invitrogen), and recombinant clones were sequenced. For PCR withspecific primers, either genomic DNA or first-strand cDNA reversetranscribed from RNA was subjected to 25 to 35 cycles of amplificationusing incubations at 94° C. for 45 sec, 60° C. for 45 sec and 72° C. for2 or 3 min. The amplified DNA was separated on an agarose gel. DNA bandswere subcloned into pcDNA3 (Invitrogen) for expression studies andsequence analysis, or subjected to Southern anlaysis for the comparativereverse transcription polymerase chain reaction (RT-PCR) assay(described herein below).

A human genomic library in EMBL-3 SP6/T7 (Clontech) was plated andtransferred to Colony Plaque Screen filters (New England Nuclear). Thefilters were screened under conditions of either high or reducedstringency. High stringency consisted of overnight hybridization in 50%formamide, 1M sodium chloride, 1% SDS, 10% dextran sulfate, 100 μg/mldenatured salmon sperm at 42° C., with filters being washed in 2x SSC,1% SDS at 65° C. for 1 hr. Reduced stringency consisted of the samehydridization solution at 42° C., except the formamide concentration was25%; the filters were washed in 2x SSC, 1% SDS at 55° C. for 1 hr.Lambda phage that hybridized to the probe were plaque-purified.

A novel CDNA fragment (364 bp) was found by sequence analysis usingstandard techniques to be 60% identical at the amino acid level witheither the human Mel-1a receptor or the Xenopus melatonin receptor. ThisPCR-fragment was labeled by a standard random priming technique and usedto probe a human genomic library at high stringency. From 1×10⁶recombinants, seven positively hydridizing clones were identified andplaque purified. A 6 kb SacI-fragment of one of the genomic clones whichhybridized to the PCR-generated cDNA fragment was subcloned andpartially sequenced. This fragment contained the 3' end of the putativecoding region and extended 5' to the GN motif in the first cytoplasmicloop, in which an apparent intron occurred; a consensus intron splicesite occurs at an identical location in the human and sheep Mel-1areceptor genes (SEQ ID NO:11 and SEQ ID NO:3, respectively; Reppert, S.M., Weaver, D. R. & Ebisawa, T. (1994) Neuron 13: 1177-1185). To obtainthe 5' portion of the coding region, a 160 bp fragment encoding thefirst transmembrane domain of the sheep Mella-melatonin receptor wasused to reprobe the seven positive genomic clones at reduced stringency(Reppert, S. M. et al. (1994), supra). A 2.3 kb SacI-fragment of one ofthe genomic clones which hydridized to the sheep receptor fragmentd wassubcloned and sequenced by standard techniques. This SacI-fragmentcontained the apparent 5' end of the coding region which includes anupstream, in-frame methionine with a consensus sequence for theinitiation of translation (Kozak, M. (1987) Nudeic Acids Res. 15:8125-8148) and a consensus site for N-linked glycosylation. RT-PCR ofRNA from human brain using specific primers directed at the 5' and 3'ends of the putative coding region amplified the expected cDNA with theappropriate splicing predicted from genomic analysis, indicating thatthe putative receptor gene is transcribed. A PCR-generated construct ofthe coding region of human Mel-1b receptor was subcloned into pcDNA3 forexpression studies and sequence analysis. The deduced amino acidsequence of human Mel-1b receptor was identical with the correspondingsequence of the SacI-genomic fragments.

Human melatonin-1b receptor encodes a protein of 362 amino acids (SEQ IDNO:16) with a predicted molecular mass of 40,188, not includingposttranslational modifications (FIG. 6). Human Mel-1b is a member of anewly described melatonin receptor group that is distinct from the otherreceptor groups (e.g., biogenic amine, neuropeptide, and photopigmentreceptors) that comprise the prototypic G protein-coupled receptorfamily (Ebisawa, et al. (1994) Proc. Natl. Acad Sci. USA 91, 6133-6137;Reppert, S. M. et al. (1994), supra). Unique features of this groupinclude a NRY motif just downstream from the third transmembrane domain(rather than DRY) and a NAXXY motif (SEQ ID NO:17) in transmembrane 7(rather than NPXXY (SEQ ID NO:7)) (FIG. 18). In addition, the humanMel-1b receptor, the mammalian Mel-1a receptors, and the Xenopusmelatonin receptor all have a CYICHS motif (SEQ ID NO:18) immediatelydownstream from NRY in the third cytoplasmic loop which is a consensussite for cytochrome c family heme binding (Mathews, F. S. (1985) Prog.Biophys. Mol. Biol. 45: 1-56). Pair-wise comparisons of the human Mel-1breceptor, the human Mel-1a receptor and the Xenopus melatonin receptorreveal approximately 60% amino acid identity for any pair of the threesequences (FIG. 18). Within the transmembrane domains the amino acididentity among any two of the three sequences is 73%. The mostdissimilar regions among any two of the three receptors are in theamino- and carboxy-terminal regions and in the second and thirdcytoplasmic loops. Within the amino terminus there is one consensus sitefor N-linked glycosylation for the Xenopus melatonin receptor and thehuman Mel-1b receptor, while there are two sites in the amino terminusof the human Mel-1a receptor (FIG. 18, lower). The possibility ofadditional upstream translation start sites cannot be excluded.

Binding Studies of the Recombinant Human High-Affinity Mel-1b Receptor

Binding and pharmacological properties of the human Mel-1b receptor wereexamined by transiently expressing the receptor CDNA in COS-1 cells.

Expression studies were performed as follows. COS-1 and NIH 3T3 cellswere grown as monolayers in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% fetal calf serum, penicillin (50 U/ml), andstreptomycin (50 μg/ml), in 5% CO₂ at 37° C. For ligand binding studies,melatonin receptor cDNAs in pcDNA3 were introduced into COS-1 cellsusing the DEAE-dextran method (Cullen, B. (1987) Methods Enzymol. 152,684-704). Three days after transfection, medium was removed, and thedishes were washed with PBS. The cells were harvested in Hank's balancedsalt solution and centrifuged (1400×g; 10 min, 4° C.). The resultantpellets were stored at -80° C. Crude membrane homogenates were preparedby thawing the pellets on ice and resuspending them in TME buffer (pH7.4) consisting of 50 mM Tris base, 12.5 mM MgCl2, 1 mM EDTA, andsupplemented with 10 μg/ml aprotinin and leupeptin, and 100 μMphenylmethylsulfonylfluoride. The cells were then homogenized using adounce homogenizer and centrifuged (45,000×g; 15 min at 4° C.). Theresulting pellet was resuspended with a dounce homogenizer in. TME andfrozen at 80° C. in aliquots.

Binding assays were performed in duplicate in a final volume of 200 μl,consisting of 20 μl radioligand, 20 μl TME containing either melatoninor displacer, and 160 μl membrane homogenates. Incubations wereinitiated by the addition of the membrane preparation and were conductedat 37° C. for 60 min. Nonspecific binding was defined by 10 μMmelatonin. All determinations were done in either duplicate ortriplicate.

Protein measurements were performed by the method of Bradford (Bradford,M. M. (1976) Anal. Biochem. 72, 248-254), using bovine serum albumin asthe standard. Binding data were analyzed by computer using the LIGANDProgram of Munson and Rodbard (Munson, P. J. and Rodbard, D. (1980)Anal. Biochem. 107, 220-239).

For comparison, binding and pharmacology of COS-1 cells transientlyexpressing the human Mel-1a receptor were assessed in parallel.Scatchard transformation of the saturation data showed that COS-1 cellstransfected with either receptor bind ¹²⁵ I-Mel with high affinity. TheK_(d) of human Mel-1b receptor was 1.6±0.3×10⁻¹ M (mean+SE; n=5experiments) (FIG. 19). This value represents a 4-fold lower affinitythan that of the human Mel-1a receptor (K_(d) =6.5±0.6×10⁻¹¹ M; n=3)found in parallel experiments. The B_(max) values were 2.7±0.1 pmol/mgmembrane protein for human Mel-1b receptor and 2.8±0.4 pmol/mg membraneprotein for the human Mel-1a receptor. The pharmacologicalcharacteristics for inhibition of specific ¹²⁵ IMel binding in acutelytransfected COS-1 cells were next examined for Mel-1b receptor andcompared with those of the human Mel-1a receptor (FIG. 20; Table 1).

                  TABLE 1    ______________________________________    Competition of various ligands for specific .sup.125 I-Me1 binding    in COS-1 cells transfected with either human Me1-1b or the    Me1-1a receptor cDNA               K.sub.i  (nM)     Ratio    Compound   Me1-1b     Me1-1a     (Me1-1a/Me1-1b)    ______________________________________    2-iodomelatonin               0.17 ± 0.02                          0.09 ± 0.01                                     0.5    2-phenylmelatonin               0.26 ± 0.06                          0.21 ± 0.06                                     0.8    S20098     0.23 ± 0.04                          0.72 ± 0.11                                     3.1    6-chloromelatonin               0.66 ± 0.04                          6.78 ± 0.91                                     10.3    melatonin  1.11 ± 0.13                          1.48 ± 0.21                                     1.3    NAS        595 ± 127                          986 ± 137                                     1.6    5-HT       >10,000    >10,000    --    prazosin   >10,000    >10,000    --    ______________________________________     K.sub.i  values are mean ± SE of 3-5 experiments for each drug. NAS:     Nacetyl-5-hydroxytryptamine. 5HT: 5hydroxytryptamme. S20098, a melatonin     analog was obtained from BristolMyers Squibb, Princeton, NJ.

For human Mel-1b, the rank order of inhibition of specific ¹²⁵ I-Melbinding by six ligands was2-iodomelatonin>2-phenylmelatonin>S-20098>6-chloromelatonin>melatonin>N-acetyl-5-hydroxytryptamine(FIG. 20a; Table 1). Micromolar concentrations of prazosin or5-hydroxytryptamine did not inhibit specific ¹²⁵ I-Mel binding. The rankorder of inhibition of specific ¹²⁵ I-Mel binding for human Mel-1breceptor was very similar to that found in parallel experiments for thehuman Mel-1a melatonin receptor, except that 6chloromelatonin was10-fold more potent in inhibiting specific ¹²⁵ I-Mel binding in cellsexpressing human Mel-1b receptor (FIG. 20b; Table 1). Thus, human Mel-1breceptor cDNA encodes a protein with ¹²⁵ I-Mel binding characteristicsthat are quite similar to those of the Mel-1a melatonin receptor.

Melatonin Inhibits cAMP Accumulation in Mel-1b-expressing Cells.

The recombinant Mel-1b receptor is coupled to inhibition of adenylylcyclase as is the Mel-1a melatonin receptor (Reppert, S. M. et al.(1994), supra).

For these studies, we used clonal lines of NIH 3T3 cells stablytransfected with the receptor cDNA in pcDNA3. COS-1 and NIH 3T3 cellswere grown as monolayers in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% fetal calf serum, penicillin (50 U/ml), andstreptomycin (50 μg/ml), in 5% CO₂ at 37° C.

For ligand binding studies, melatonin receptor cDNAs in pcDNA3 wereintroduced into COS-1 cells using the DEAE-dextran method (Cullen, B.(1987) supra). Three days after transfection, medium was removed, andthe dishes were washed with PBS. The cells were harvested in Hank'sbalanced salt solution and centAfuged (1400×g; 10 min, 4° C.). Theresultant pellets were stored at -80° C. Crude membrane homogenates wereprepared by thawing the pellets on ice and resuspending them in TMEbuffer (pH 7.4) consisting of 50 mM Tris base, 12.5 mM MgCl2, 1 mM EDTA,and supplemented with 10 μg/ml aprotinin and leupeptin, and 100 μMphenylmethylsulfonylfluoride. The cells were then homogenized using adounce homogenizer and centrifuged (45,000×g; 15 min at 4° C.). Theresulting pellet was resuspended with a dounce homogenizer in TME andfrozen at -80° C. in aliquots. Binding assays were performed induplicate in a final volume of 200 μl, consisting of 20 μl radioligand,20 μl TME containing either melatonin or displacer, and 160 μl membranehomogenates. Incubations were initiated by the addition of the membranepreparation and were conducted at 37° C. for 60 min. Nonspecific bindingwas defined by 10 μM melatonin. All determinations were done in eitherduplicate or triplicate. Protein measurements were performed by themethod of Bradford (Bradford, M. M. (1976) supra), using bovine serumalbumin as the standard. Binding data were analyzed by computer usingthe LIGAND Program of Munson and Rodbard (Munson, P. J. & Rodbard, D.(1980) supra). For cAMP studies, the receptor cDNA in pcDNA3 wasintroduced into NIH 3T3 cells using Lipofectamine (GIBCO/BRL).Transformed NIH 3T3 cells resistant to Geneticin, G418 (at 1.0 mg/ml;Gibco/BRL) were isolated and single colonies expressing melatoninreceptor binding (>200 fmol/mg total cellular protein) were isolated.

Transformed NIH 3T3 cells were plated in triplicate on 35 mm dishes.Forty-eight hours later, the cells were washed (2X) with DMEM andpreincubated with 250 uM 3-isobutyl-1-methylxanthine (IBMX) in DMEM for10 min at 37° C. Cells were then incubated with or without drugs in DMEMwith 250 AM IBMX for 10 min at 37° C. At the end of treatment, themedium was aspirated and 0.5 ml of 50 mM acetic acid was added. Thecells were collected, transferred to an Eppendorf tube, boiled for 5min, and centrifuged (13,750 rpm for 15 min). The supernatant wascollected and assayed for cAMP. All determinations were done intriplicate. Cyclic AMP levels were determined in duplicate byradioimmunoassay (New England Nuclear). ¹²⁵ I-Mel was purchased from NewEngland Nuclear. All drugs used in competition studies were purchasedfrom Sigma, Research Biochemicals or were synthesized by standardmethods. All other chemicals were purchased from Sigma.

Results of these studies showed that melatonin (1 μM) did not increasebasal cAMP levels in stably transfected NIH 3T3 cells. Melatonin didcause a dose-dependent inhibition of the increase in cAMP accumulationinduced by 10 μM forskolin (FIG. 21); the maximal inhibition of the meanforskolin stimulated cAMP value was at 10⁻⁸ M melatonin. The estimatedIC₅₀ value of this response (ca. 1×10⁻⁹ M) was very similar to thecomputer generated K_(i) value (1.11±0.13×10⁻⁹ M) determined formelatonin inhibition of specific ¹²⁵ I-Mel binding (FIG. 20; Table 1).Thus, the recombinant melatonin-1b receptor is negatively coupled to thecAMP regulatory system.

Characteristics of the Human High Affinity Mel-1b Receptor Gene and itsExpression

Restriction endonuclease mapping and PCR analysis of genomic clonesshowed that the portion of the gene that encodes the coding region ofhuman Mel-1b receptor is comprised of two exons, separated by an intronthat is approximately 9.0 kb in length. Southern analysis of humangenomic DNA digested with several different restriction endonucleaseswas performed using a PCR-fragment of the second exon of human Mel-1bDNA as a hybridization probe. Under high stringency conditions, apattern of single bands was observed, suggesting that human Mel-1breceptor is encoded by a single copy gene.

To localize the gene for human Mel-1b, an intronic PCR assay wasdeveloped that would amplify only the human Mel-1b receptor gene. Apanel of 43 human-rodent somatic cell hybrids that contained definedoverlapping subsets of human chromosomes was screened (Geissler, E. N.,Liao, M., Brook, J. D., Martin, F. H., Zsebo, K. M., Housman, D. E. &Galli, S. J. (1991) Somatic Cell Genet. 17, 207-214; Pelletier, J.,Brook, D. J. & Housman, D. E. (1991) Genomics 10, 1079-1082; NIGMSMapping Panel #2, Coriell Institute, Camden, N.J.). Using primer5'-CTGTGCCTCTAAGAGCCACTTGGTTTC-3' (SEQ ID NO:19) and primer5'TATTGAAGACAGAGCCGATGACGCTCA3' (SEQ ID NO:29), PCR amplified a singleband only in those cell lines containing human chromosome 11. The Mel-1breceptor gene was further localized to band 11q21-22 by PCR screening ofa panel of somatic cell hybrids containing various deletion fragments ofhuman chromosome 11 (Glaser, T., Housman, D., Lewis, W. H., Gerhard, D.& Jones, C. (1989) Somat. CelI. Mol. Genet. 15, 477-501; FIG. 23). Thegene encoding human Mel-1b receptor has been given the designation MTNR1B.

To assess the tissue distribution of human Mel-1b mRNA, comparativeRT-PCR analysis was performed using a modification of a previouslydescribed procedure (Kelly, M. R., Jurgens, J. K., Tentler, J.,Emanuele, N. V., Blutt, S. E., Emanuele, M. A. (1993) Alcohol 10:185-189). Poly(A)⁺ RNA was purchased from Clontech and 2 μg from eachtissue was primed with random hexamers and reverse transcribed aspreviously described (Reppert, S. M., Weaver D. R., Stehle, J. H. &Rivkees, S. A. (1991) Mol. Endocrinol. 5:1037-1048). The cDNA wassubjected to 25 cycles of amplification with 200 nm each of two specificprimers.

The Mel-1b and Mel-1a receptor primers were designed so that they wouldamplify cDNA across the intron splice sites in the first cytoplasmicloop. Since the introns for both the Mel-1b and Mel-1a receptor genesare large (>8 kb), amplification of the appropriate sized cDNA fragmentswould eliminate the possibility of amplification of genomic DNA. Thehuman Mel-1b receptor primers were 5'-TCCTGGTGATCCTCTCCGTGCTCA-3' (SEQID NO:20) and 5'-AGCCAGATGAGGCAGATGTGCAGA-3' (SEQ ID NO:21), andamplified a band of 321 bp. The Mel-1a receptor primers were5'-TCCTGGTCATCCTGTCGGTGTATC-3' (SEQ ID NO:22) and5'-CTGCTGTACAGTTTGTCGTACTTG-3' (SEQ ID NO:23), and amplified a band of285 bp. Histone-H3.3 served as a control to verify the amount oftemplate for each sample. The histone H3.3 primers were5'-GCAAGAGTGCGCCCTCTACTG-3' (SEQ ID NO:24) and5'-GGCCTCACTTGCCTCCTGCAA-3' (SEQ ID NO:25), and amplified a band of 217bp.

After PCR, the reaction products were subjected to electrophoresisthrough a 1.5% agarose gel and blotted onto GeneScreen (New EnglandNuclear). To increase the specificity of the assay, blots werehybridized with 25-mer oligonucleotides, labeled with γ-32P!ATP by T4polynucleotide kinase. For each primer pair, the oligonucleotide probeswere specific for a sequence of the amplified fragment between theprimers. Oligonucleotide sequences were 5'-CTAATCCTCGTGGCCAATCTTCTATG-3'(SEQ ID NO:26) for human Mel-1b receptor;5'-TTGGTGCTGATGTCGATATTTAACA-3' (SEQ ID NO:27) for the human Mel-1areceptor; and 5'-CACTGAACTTCTGATTCGCAAACTT-3' (SEQ ID NO:28) forhistoneH3.3. Hybridizing conditions were 45° C. overnight in 0.5M NaPO₄(pH 7.2), 7% SDS, 1% BSA and 1 mM EDTA. The blots were washed twice in0.2M NaPO₄, 1% SDS and 1 mM EDTA at 45° C. for 30 min.

A 364 bp fragment of the rat homolog of the human Mel-1b receptor cDNAwas cloned by RT-PCR from rat brain RNA; the rat cDNA fragment was 81%identical at the amino acid level with human Mel-1b receptor. The ratfragment was used to probe a Northern blot containing 5 μg poly(A)⁺ RNAfrom each of 20 different rat tissues. No positive hybridization signalswere found. Furthermore, In situ hybridization using an antisense cRNAprobe to the rat fragment did not reveal a hybridization signal in PT orSCN, sites which gave a positive hybridization signal in the same insitu run using an antisense cRNA probe to the Mel-1a receptor (Reppert,S. M., Weaver, D. R. & Ebisawa, T. (1994) Neuron 13, 1177-1185).

Because of the apparent low level of receptor transcripts, a comparativeRT-PCR assay was used to examine the expression of human Mel-1b andMel-1a receptor genes in 6 human tissues (FIG. 22). Human Mel-1breceptor was expressed in retina, with much lower expression in wholebrain and hippocampus. The human Mel-1a receptor was clearly expressedin whole brain, with just detectable expression in retina andhippocampus. Neither Mel-1b nor Mel-1a receptor mRNA was detected inpituitary, liver of spleen. To ensure consistency in the amount of RNAreverse transcribed and the efficiency of the reverse transcriptionreactions among the tissues examined, the histone H3.3 cDNA wasamplified from each tissue examined; very comparable amplificationsoccurred among the six tissues (FIG. 22).

Relative Characteristics of the Human High Affinity Mel-1a and Mel-1bReceptors

One feature that distinguishes the Mellb-receptor from the Mel-1areceptor is its tissue distribution. The substantially greaterexpression of the Mel-1b receptor in retina suggests that melatonin mayexert its effects on mammalian retinal physiology through this receptor.Melatonin inhibits the Ca⁺² -dependent release of dopamine in rabbitretina through activation of receptors with pharmacologic specificitycomparable with that reported here for the Mel-1b receptor (Dubocovich,M. L. & Takahashi, J. (1987) Proc. Natl. Acad. Sci. USA 84, 3916-3920;Dubocovich, M. L. (1983) Nature 306, 782-4). Melatonin appears to act inthe retina to affect several light-dependent functions, includingphotopigment disc shedding and phagocytosis (Besharse, J. C. & Dunis, D.A. (1983) Science 219:1341-1343; Cahill, G. M., Grace, M. S. & Besharse,J. C. (1991) Cell. Mol. Neurobiol. 11:529-560).

The discovery of the Mel-1b receptor which has similar binding andfunctional characteristics to those of the Mel-1a receptor make itconceivable that the Mel-1b receptor also participates in the circadianand/or reproductive actions of melatonin. Even though Mel-1b receptormRNA is not detectable by in situ hybridization in rat SCN or PT, it maybe present and functional in these or other neural sites at levels notdetectable using standard detection methods.

A second distinguishing feature of the Mel-1b receptor is its chromosomelocation. The Mel-1b melatonin receptor maps to human chromosome11q21-22, a region syntenic to mouse chromosome 9 in the region of theD₂ -dopamine receptor (Drd2) and thymus cell antigen 1 (Thy1) loci(Goldsborough et al. (1993) Nucl. Acids Res. 21:127-132; Seldin, M. F.,Saunders, A. M., Rochelle, J. M. and Howard, T. A. (1991) Genomics9:678-685). This contrasts with the Mel-1a receptor which maps to humanchromosome 4q35.1 and mouse chromosome 8. Thus, these two structurallyand functionally related melatonin receptors did not merely evolve bysimple tandem duplication of an ancestral gene, but suggests that othermechanisms, such as chromosomal rearrangement and duplication, wereinvolved.

The discovery of a new member of the G protein-coupled, melatoninreceptor family shows that at least two distinct genes have evolved tosubserve melatonin's functions. The development of a method ofidentifying pharmacological agents which selectively affect Mel-1a andMel-1b receptor function is an important therapeutic application madeavailable by the disclosed invention.

Relative Characteristics of the Xenopus and Mammalian Melatonin-1aHigh-Affinity Receptors

Acute transfection of COS-7 cells with the Xenopus melatonin receptorand the sheep Mel-1a receptor clones results in transient expression ofreceptors that bind ¹²⁵ I-melatonin with high affinity (FIG. 9 and FIG.12b). Additionally, specific ¹²⁵ I-melatonin binding to Xenopus receptortransiently expressed in cells is inhibited by six ligands in a rankorder that is identical to that reported for the endogenous Mel-1areceptor in reptiles, birds, and mammals (FIG. 9) (Dubocovich et al.(1987), supra; Rivkees et al. (1989), supra; Morgan, P. J. et al. (1989)supra). The ability of the recombinant Xenopus high-affinity melatoninreceptor to inhibit the forskolin-stimulated increase in cAMPaccumulation in stably transfected CHO cells is consistent with studiesof the endogenous receptor which show that a major signal transductionpathway for the high-affinity Mel -1a receptor is inhibition of adenylylcyclase (Abe, K. et al. (1969), supra; White et al. (1987), supra).Finally, Xenopus melatonin receptor mRNA is moderately expressed in thecells whose RNA was used to generate the cDNA library. Thus, the clonedreceptor likely mediates the potent effects of melatonin on pigmentaggregation in frog melanophores. Structurally, the protein encoded bythe melatonin receptor cDNA defines a new receptor group within thelarge superfamily of G protein-coupled receptors.

Previous studies using quantitative ¹²⁵ I-Mel autoradiography in thehuman SCN have generally shown high affinity for melatonin and6-chloromelatonin and very low affinity for serotonin (Reppert et al.,(1988) supra), all consistent with the pharmacological characteristicsof the recombinant human receptor (FIG. 15). The pharmacologicalcharacteristics of the recombinant sheep Mel-1a receptor are virtuallyidentical to those of the endogenous melatonin la receptor in sheep PT(Morgan et al., J. Endocrinol. (1989) 1:1-4). The difference between thesheep and human Mel-1a receptors in their affinities for6-chloromelatonin is reproducible and equally apparent when the sheepand human Mel-1a receptors are examined in the same assay run.

Polypeptide Expression

Polypeptides according to the invention may be produced bytransformation of a suitable host cell with all or part of ahigh-affinity melatonin receptor-encoding cDNA fragment (e.g., the cDNAsdescribed above) in a suitable expression vehicle, and expression of thereceptor.

Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used to provide therecombinant receptor protein. The precise host cell used is not criticalto the invention. The receptor may be produced in a prokaryotic host(e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiaeor mammalian cells, e.g., COS-6M, COS-7 NIH/3T3, or Chinese HamsterOvary cells). Such cells are available from a wide range of sources(e.g., the American Type Culture Collection, Rockville, Md.). The methodof transfection and the choice of expression vehicle will depend on thehost system selected. Transformation and mammalian cell transfectionmethods are described, e.g., in Ausubel et al. (Current Protocols inMolecular Biology, John Wiley & Sons, New York, (1989)); expressionvehicles may be chosen from those provided, e.g., in Cloning Vectors: ALaboratory Manual (Pouwels, P. H. et al., (1985), Supp. 1987).

One particularly preferred expression system is the Chinese hamsterovary (CHO) cell (ATCC Accession No. CCL 61) transfected with apcDNAI/NEO expression vector (InVitrogen, San Diego, Calif.). pcDNAI/NEOprovides an SV40 origin of replication which allows replication inmammalian systems, a selectable neomycin gene, and SV40 splicing andpolyadenylation sites. DNA encoding the human, sheep, or Xenopushigh-affinity melatonin receptor or an appropriate receptor fragment oranalog (as described above) would be inserted into the pcDNAI/NEO vectorin an orientation designed to allow expression. Other preferable hostcells which may be used in conjunction with the pcDNAI/NEO expressionvehicle include NIH/3T3 cells (ATCC Accession No. 1658). The expressionmay be used in a screening method of the invention (described below) or,if desired, the recombinant receptor protein may be isolated asdescribed below.

Alternatively, the high-affinity melatonin receptor (or receptorfragment or analog) is expressed by a stably-transfected mammalian cellline.

A number of vectors suitable for stable transfection of mammalian cellsare available to the public, e.g., see Pouwels et al. (supra); methodsfor constructing such cell lines are also publicly available, e.g., inAusubel et al. (supra). In one example, cDNA encoding the receptor (orreceptor fragment or analog) is cloned into an expression vector whichincludes the dihydrofolate reductase (DHFR) gene. Integration of theplasmid and, therefore, the high-affinity melatonin receptor-encodinggene into the host cell chromosome is selected for by inclusion of0.01-300 μM methotrexate in the cell culture medium (as described inAusubel et al., supra). This dominant selection can be accomplished inmost cell types. Recombinant protein expression can be increased byDHFR-mediated amplification of the transfected gene. Methods forselecting cell lines bearing gene amplifications are described inAusubel et al. (supra); such methods generally involve extended culturein medium containing gradually increasing levels of methotrexate.DHFR-containing expression vectors commonly used for this purposeinclude pCVSEII-DHFR and pAdD26SV(A) (described in Ausubel et al.,supra). Any of the host cells described above or, preferably, aDHFR-deficient CHO cell line (e.g., CHO DHFR cells, ATCC Accession No.CRL 9096) are among the host cells preferred for DHFR selection of astably-transfected cell line or DHFR-mediated gene amplification.

One particularly preferred stable expression system is a CHO cell (ATCC)stably transfected with a pcDNAI/NEO (InVitrogen, San Diego, Calif.)expression vector.

Expression of the recombinant receptor (e.g., produced by any of theexpression systems described herein) may be assayed by immunologicalprocedures, such as Western blot or immunoprecipitation analysis ofrecombinant cell extracts, or by immunofluorescence of intactrecombinant cells (using, e.g, the methods described in Ausubel et al.,supra). Recombinant receptor protein is detected using an antibodydirected to the receptor. Described below are methods for producinghigh-affinity melatonin receptor antibodies using, as an immunogen, theintact receptor or a peptide which includes a suitable high-affinitymelatonin receptor epitope. To detect expression of a high-affinitymelatonin receptor fragment or analog, the antibody is preferablyproduced using, as an immunogen, an epitope included in the fragment oranalog.

Once the recombinant high-affinity melatonin receptor protein (orfragment or analog, thereof) is expressed, it is isolated, e.g., usingimmunoaffinity chromatography. In one example, an anti-high-affinitymelatonin receptor antibody may be attached to a column and used toisolate intact receptor or receptor fragments or analogs. Lysis andfractionation of receptor-harboring cells prior to affinitychromatography may be performed by standard methods (see, e.g., Ausubelet al., supra). Once isolated, the recombinant protein can, if desired,be further purified, e.g., by high performance liquid chromatography(see, e.g., Fisher, Laboratory Techniques In Biochemistry And MolecularBiology, eds., Work and Burdon, Elsevier, (1980)).

Receptors of the invention, particularly short receptor fragments, canalso be produced by chemical synthesis (e.g., by the methods describedin Solid Phase Peptide Synthesis, (1984) 2nd ed., The Pierce ChemicalCo., Rockford, Ill.).

Assays for High-Affinity Melatonin Receptor Function

Useful receptor fragments or analogs in the invention are those whichinteract with melatonin. Such an interaction may be detected by an invitro functional assay (e.g., the cAMP accumulation assay describedherein). This assay includes, as components, forskolin for induced cAMPaccumulations, melatonin, and a recombinant high-affinity melatoninreceptor (or a suitable fragment or analog) configured to permitmelatonin binding (e.g., those polypeptides described herein). Melatoninand forskolin may be obtained from Sigma (St. Louis, Mo.) or similarsupplier.

Preferably, the high-affinity melatonin receptor component is producedby a cell that naturally presents substantially no receptor on itssurface, e.g., by engineering such a cell to contain nucleic acidencoding the receptor component in an appropriate expression system.Suitable cells are, e.g., those discussed above with respect to theproduction of recombinant receptor, such as CHO cells or COS-7 cells.

Screening For High-Affinity Melatonin Receptor Antagonists and Agonists

As discussed above, one aspect of the invention features screening forcompounds that antagonize the interaction between melatonin and thehigh-affinity melatonin receptor, thereby preventing or reducing thecascade of events that are mediated by that interaction. The elements ofthe screen are forskolin to induce intracellular accumulation of cAMP,melatonin, and recombinant high-affinity receptor (or a suitablereceptor fragment or analog, as outlined above) configured to permitdetection of melatonin function. As described above, melatonin andforskolin may be purchased from Sigma, and a full-length sheep Mel-1areceptor or Xenopus high-affinity elatonin receptor, or a humanhigh-affinity melatonin 1a or 1b receptor (or a melatonin-bindingfragment or analog of the Xenopus, sheep or human receptors) may beproduced as described herein. Preferably, such a screening assay iscarried out using cell lines stably transfected with the high-affinitymelatonin receptor. Most preferably, the untransfected cell linepresents substantially no receptor on its cell surface.

Activation of the heterologous high-affinity melatonin receptor withmelatonin or an agonist (see above) leads to reduction of intracellularcAMP concentration, providing a convenient means for measuring melatoninor agonist activity. Such an agonist may be expected to be a usefultherapeutic agent for circadian rhythm disorders such as jet lag,day/night cycle disorders in humans or mating cycle alterations inanimals such as sheep. Appropriate candidate agonists include melatoninanalogs or other agents which mimic the action of melatonin.

Inclusion of potential antagonists in the screening assay along withmelatonin allows for the screening and identification of authenticreceptor antagonists as those which decrease melatonin-mediatedintracellular cAMP reduction. Receptor bearing cells incubated withforskolin (for initial induction cAMP concentration) or melatonin(alone, i.e., in the absence of inhibitor) are used as a "control"against which antagonist assays are measured.

Appropriate candidate antagonists include high-affinity melatoninreceptor fragments, particularly, fragments of the protein predicted tobe extracellular (see FIG. 7) and therefore likely to bind melatonin;such fragments would preferably including five or more amino acids.Other candidate antagonists include melatonin analogs as well as otherpeptide and non-peptide compounds and anti-high-affinity melatoninreceptor antibodies.

Another aspect of the invention features screening for compounds thatact as high-affinity melatonin receptor agonists; such compounds areidentified as those which bind a high-affinity melatonin receptor andmimic the cascade of events that are normally mediated by thatinteraction. This screen requires recombinant cells expressingrecombinant high-affinity melatonin receptor (or a suitable receptorfragment or analog, as outlined herein) configured to permit detectionof high-affinity melatonin receptor function. In one example, acandidate agonist is added to CHO cells stably expressing recombinantreceptor and intracellular cAMP levels are measured (as describedabove). An agonist useful in the invention is one which imitates thenormal melatonin-mediated signal transduction pathway leading, e.g., toan decrease in intracellular cAMP concentration.

Appropriate candidate agonists include melatonin analogs or otherchemical agents capable of mimicking the action of melatonin.

Preparation of a Transgenic Animal Containing Recombinant Melatonin-1aand/or Melatonin-1b Genes

There are several means by which transgenic animals can be made. Atransgenic animal (such as a mammal) may be constructed by one ofseveral techniques, including targeted insertion of an exogenousmelatonin receptor gene into the endogenous gene of the animal, or othermethods well known to those skilled in the art.

A transgenic mammal whose germ cells and somatic cells contain anexogenous melatonin-1a or melatonin-1b receptor gene is produced bymethods known in the art. See, for example, U.S. Pat. No. 4,736,866describing production of a transgenic mammal, herein incorporated byreference. Generally, the DNA sequence encoding an exogenousmelatonin-1a or -1b receptor gene is introduced into the animal, or anancestor of the animal, at an embryonic stage (preferably the one-cell,or fertilized oocyte, stage, and generally not later than about the8-cell stage). There are several methods known to the art of introducinga foreign gene into an animal embryo to achieve stable expression of theforeign gene. One method is to transfect the embryo with the gene as itoccurs naturally, and select transgenic animals in which the foreigngene has integrated into the genome at a locus which results in itsexpression. Other methods involve modifying the foreign gene or itscontrol sequences prior to introduction into the embryo. For example,the melatonin-1a or -1b receptor gene may be modified with an enhanced,inducible, or tissue-specific promoter.

Tissues of transgenic mammals are analyzed for the presence of exogenousmelatonin-1a or -1b receptor, either by directly analyzing mRNA, or byassaying the tissue for exogenous melatonin-1a or -1b receptor.

Using the Transgenic Mammal to Determine Melatonin Agonist- orAntagonist-Related Effects

The animals described above can be used to determine whether candidatecompounds are melatonin antagonists or agonists for the Mel-1a or Mel-1breceptors.

Assessing Melatonin Agonists or Antagonists in vivo

One aspect of the invention features screening for compounds thatagonize or antagonize melatonin activity in vivo. The elements of thescreen are a Mel-1a or Mel-1b transgenic mammal and a potentialmelatonin agonist or antagonist in a suitable formulation foradministration to the mammal. Detection of a change in the phenotype ofinterest (e.g., sleep/wake cycle or reproductive cycle) relative to acontrol transgenic mammal to which no agonist or antagonist has beenadministered indicates a potentially useful candidate compound.

Anti-High-Affinity Melatonin Receptor Antibodies

High-affinity melatonin receptor (or immunogenic receptor fragments oranalogs) may be used to raise antibodies useful in the invention. Asdescribed above, receptor fragments preferred for the production ofantibodies are those fragments deduced or shown experimentally to beextracellular.

Antibodies directed to high-affinity melatonin receptor peptides areproduced as follows. Peptides corresponding to all or part of theputative extracellular loops or the extracellular N-terminal domain areproduced using a peptide synthesizer, by standard techniques. Thepeptides are coupled to KLH with m-maleimide benzoic acidN-hydroxysuccinimide ester. The KLH-peptide is mixed with Freund'sadjuvant and injected into animals, e.g. guinea pigs or goats, toproduce polyclonal antibodies. Monoclonal antibodies may be preparedusing the high-affinity melatonin polypeptides described above andstandard hybridoma technology (see, e.g., Kohler et al., Nature (1975)256:495, 1975; Kohler et al., Eur. J. Immunol. (1976) 6:292; Kohler etal., Eur. J. Immunol. (1976) 6:511; Hammerling et al., in MonoclonalAntibodies and T Cell Hybridomas, Elsevier, N.Y., (1981); and Ausubel etal., supra). Antibodies are purified by peptide antigen affinitychromatography.

Once produced, antibodies are tested for specific high-affinitymelatonin receptor recognition by Western blot or immunoprecipitationanalysis (by the methods described in Ausubel et al., supra).

Antibodies which specifically recognize the high-affinity melatoninreceptor are considered to be likely candidates for useful antagonists;such candidates are further tested for their ability to specificallyinterfere with the interaction between melatonin and its receptor (usingthe functional antagonist assays described herein). Antibodies whichantagonize melatonin: high-affinity melatonin receptor binding orhigh-affinity melatonin receptor function are considered to be useful asantagonists in the invention.

Therapy

Particularly suitable therapeutics for the treatment of circadian rhythmdisorders in humans as well as for regulating changes in thereproductive cycle of seasonally breeding animals are the agonists andantagonists described above formulated in an appropriate buffer such asphysiological saline. Where it is particularly desirable to mimic areceptor fragment conformation at the membrane interface, the fragmentmay include a sufficient number of adjacent transmembrane residues. Inthis case, the fragment may be associated with an appropriate lipidfraction (e.g., in lipid vesicles or attached to fragments obtained bydisrupting a cell membrane). Alternatively, anti-high-affinity melatoninreceptor antibodies produced as described above may be used as atherapeutic. Again, the antibodies would be administered in apharmaceutically-acceptable buffer (e.g., physiological saline). Ifappropriate, the antibody preparation may be combined with a suitableadjuvant.

The therapeutic preparation is administered in accordance with thecondition to be treated. Ordinarily, it will be administeredintravenously, at a dosage, of a duration, and with the appropriatetiming to elicit the desired response. Appropriate timing refers to thetime in the natural circadian rhythm at which administration oftherapeutic preparation elicits the desired response. Alternatively, itmay be convenient to administer the therapeutic orally, nasally, ortopically, e.g., as a liquid or a spray. Again, the dosages are asdescribed above. Treatment may be repeated as necessary for alleviationof disease symptoms.

High-affinity melatonin receptor agonists can be used to reentrain theendogenous melatonin rhythm of humans; alleviate jet lag symptoms inhumans; phase shift the sleep/wake cycle of some blind people, reinforceentrainment of endogenous melatonin rhythm using low intensitylight/dark cycle; control ovulation in humans; and alter reproductivecycles in seasonally breeding animals. Antagonists may be useful incontrolling the initiation or timing of puberty in humans.

The methods of the invention may be used to screen therapeutic receptoragonists and antagonists for their effectiveness in reducingintracellular cAMP production in vitro; in altering circadian rythmn; orin altering reproductive cycles by the assays described above. Where anon-human mammal is treated or where a therapeutic for a non-humananimal is screened, the high-affinity melatonin receptor or receptorfragment or analog or the antibody employed is preferably specific forthat species.

Other Embodiments

Polypeptides according to the invention include any high-affinitymelatonin receptors (as described herein). Such receptors may be derivedfrom any source, but are preferably derived from a vertebrate animal,e.g., a human, a sheep, or a frog. These polypeptides are used, e.g., toscreen for antagonists which disrupt, or agonists which mimic, amelatonin:receptor interaction (see above).

Polypeptides of the invention also include any analog or fragment of ahigh-affinity melatonin receptor capable of interacting with melatonin(e.g., those derived from the high-affinity melatonin receptorextracellular domains). Such analogs and fragments may also be used toscreen for high-affinity melatonin receptor antagonists or agonists. Inaddition, that subset of receptor fragments or analogs which bindmelatonin and are, preferably, soluble (or insoluble and formulated in alipid vesicle) may be used as antagonists to reduce the amplitude of theendogenous melatonin cycle possibly providing for the induction ofpuberty in humans. The efficacy of a receptor analog or fragment isdependent upon its ability to interact with melatonin; such aninteraction may be readily assayed using high-affinity melatoninreceptor functional assays (e.g., those described herein).

Specific receptor analogs of interest include full-length or partialreceptor proteins including an amino acid sequence which differs only byconservative amino acid substitutions, for example, substitution of oneamino acid for another of the same class (e.g., valine for glycine,arginine for lysine, etc.) or by one or more non-conservative amino acidsubstitutions, deletions, or insertions located at positions of theamino acid sequence which do not destroy the receptors' ability tosignal melatonin-mediated reduction in intracellular cAMP concentration(e.g., as assayed above).

Specific receptor fragments of interest include any portion of thehigh-affinity melatonin receptor which is capable of interacting withmelatonin, for example, all or part of the extracellular domains(described above). Such fragments may be useful as antagonists (asdescribed above), and are also useful as immunogens for producingantibodies which neutralize the activity of the high-affinity melatoninreceptor in vivo (e.g., by interfering with the interaction between thereceptor and melatonin; see below).

Extracellular regions of novel high-affinity melatonin receptors may beidentified by comparison with related proteins of similar structure(e.g., other members of the G-protein-coupled receptor superfamily);useful regions are those exhibiting homology to the extracellulardomains of well-characterized members of the family.

Alternatively, from the primary amino acid sequence, the secondaryprotein structure and, therefore, the extracellular domain regions maybe deduced semi-empirically using a hydrophobicity/hydrophilicitycalculation such as the Chou-Fasman method (see, e.g., Chou and Fasman,Ann. Rev. Biochem. (1978) 47:251). Hydrophilic domains, particularlyones surrounded by hydrophobic stretches (e.g., transmembrane domains)present themselves as strong candidates for extracellular domains.Finally, extracellular domains may be identified experimentally usingstandard enzymatic digest analysis, e.g., tryptic digest analysis.

Candidate fragments (e.g., any extracellular fragment) are tested forinteraction with melatonin by the assays described herein (e.g., theassay described above). Such fragments are also tested for their abilityto antagonize the interaction between melatonin and its endogenousreceptor using the assays described herein. Analogs of useful receptorfragments (as described above) may also be produced and tested forefficacy as screening components or antagonists (using the assaysdescribed herein); such analogs are also considered to be useful in theinvention.

Other embodiments are within the claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 29    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1320 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: Coding Sequence    (B) LOCATION: 32...1291    (D) OTHER INFORMATION:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    TGCCTATCTCCCTTTGCCAGGGGGCAGAGAAATGATGGAGGTGAATAGCACT52    MetMetGluValAsnSerThr    15    TGCTTGGATTGCAGGACACCTGGTACCATACGAACAGAGCAGGATGCA100    CysLeuAspCysArgThrProGlyThrIleArgThrGluGlnAspAla    101520    CAGGACAGCGCATCTCAGGGACTCACCTCTGCCCTGGCGGTGGTTCTT148    GlnAspSerAlaSerGlnGlyLeuThrSerAlaLeuAlaValValLeu    253035    ATATTCACCATTGTTGTGGATGTCCTGGGCAATATATTGGTCATTTTG196    IlePheThrIleValValAspValLeuGlyAsnIleLeuValIleLeu    40455055    TCTGTCCTGAGGAACAAGAAGCTGCAGAATGCTGGAAATCTCTTTGTT244    SerValLeuArgAsnLysLysLeuGlnAsnAlaGlyAsnLeuPheVal    606570    GTCAGTTTGTCTATTGCCGATCTGGTTGTTGCTGTGTATCCCTATCCG292    ValSerLeuSerIleAlaAspLeuValValAlaValTyrProTyrPro    758085    GTCATTCTCATAGCTATTTTCCAGAATGGATGGACGCTTGGAAATATC340    ValIleLeuIleAlaIlePheGlnAsnGlyTrpThrLeuGlyAsnIle    9095100    CATTGTCAGATCAGTGGCTTCCTGATGGGACTCAGCGTTATTGGATCA388    HisCysGlnIleSerGlyPheLeuMetGlyLeuSerValIleGlySer    105110115    GTCTTCAACATAACAGCCATAGCTATCAACAGGTATTGCTACATCTGC436    ValPheAsnIleThrAlaIleAlaIleAsnArgTyrCysTyrIleCys    120125130135    CACAGCCTGAGATATGACAAGCTTTATAATCAAAGAAGCACCTGGTGC484    HisSerLeuArgTyrAspLysLeuTyrAsnGlnArgSerThrTrpCys    140145150    TACCTTGGCCTGACATGGATACTAACTATAATTGCAATCGTGCCAAAC532    TyrLeuGlyLeuThrTrpIleLeuThrIleIleAlaIleValProAsn    155160165    TTTTTTGTTGGATCACTACAGTATGACCCCAGGATTTTTTCTTGCACA580    PhePheValGlySerLeuGlnTyrAspProArgIlePheSerCysThr    170175180    TTTGCGCAGACAGTGAGTTCCTCATACACCATAACAGTAGTGGTGGTG628    PheAlaGlnThrValSerSerSerTyrThrIleThrValValValVal    185190195    CATTTTATAGTCCCTCTTAGTGTTGTGACATTCTGTTACTTAAGAATA676    HisPheIleValProLeuSerValValThrPheCysTyrLeuArgIle    200205210215    TGGGTTTTAGTGATCCAAGTCAAACACAGAGTTAGACAAGACTTCAAG724    TrpValLeuValIleGlnValLysHisArgValArgGlnAspPheLys    220225230    CAAAAGTTGACACAAACAGACTTGAGAAATTTCTTGACCATGTTTGTG772    GlnLysLeuThrGlnThrAspLeuArgAsnPheLeuThrMetPheVal    235240245    GTCTTTGTACTTTTTGCAGTTTGCTGGGCCCCCTTAAACTTTATCGGC820    ValPheValLeuPheAlaValCysTrpAlaProLeuAsnPheIleGly    250255260    CTTGCTGTGGCCATTAATCCGTTTCATGTGGCACCAAAGATTCCAGAA868    LeuAlaValAlaIleAsnProPheHisValAlaProLysIleProGlu    265270275    TGGCTGTTTGTTTTAAGCTATTTCATGGCCTATTTTAACAGTTGTCTC916    TrpLeuPheValLeuSerTyrPheMetAlaTyrPheAsnSerCysLeu    280285290295    AATGCTGTTATATATGGTGTGCTAAATCAAAACTTCCGCAAGGAGTAC964    AsnAlaValIleTyrGlyValLeuAsnGlnAsnPheArgLysGluTyr    300305310    AAAAGAATACTGATGTCCTTATTGACTCCAAGACTGTTGTTTCTTGAC1012    LysArgIleLeuMetSerLeuLeuThrProArgLeuLeuPheLeuAsp    315320325    ACATCTAGAGGAGGAACTGAGGGATTGAAAAGTAAGCCTTCGCCAGCT1060    ThrSerArgGlyGlyThrGluGlyLeuLysSerLysProSerProAla    330335340    GTAACCAACAACAATCAAGCAGATATGCTAGGAGAAGCAAGGTCACTG1108    ValThrAsnAsnAsnGlnAlaAspMetLeuGlyGluAlaArgSerLeu    345350355    TGGCTGAGCAGGAGAAATGGTGCGAAAATGGTGATCATCATCAGGCCA1156    TrpLeuSerArgArgAsnGlyAlaLysMetValIleIleIleArgPro    360365370375    AGAAAAGCACAAATTGCAATCATCCATCAAATATTCTGGCCTCAGAGT1204    ArgLysAlaGlnIleAlaIleIleHisGlnIlePheTrpProGlnSer    380385390    TCATGGGCAACATGCCGTCAAGACACAAAGATTACCGGAGAAGAAGAT1252    SerTrpAlaThrCysArgGlnAspThrLysIleThrGlyGluGluAsp    395400405    GGCTGCCGTGAACTGTGCAAGGACGGGATTTCCCAAAGGTGAGACCCAATG1303    GlyCysArgGluLeuCysLysAspGlyIleSerGlnArg    410415420    CACTATATCCACATTAT1320    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 420 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetMetGluValAsnSerThrCysLeuAspCysArgThrProGlyThr    151015    IleArgThrGluGlnAspAlaGlnAspSerAlaSerGlnGlyLeuThr    202530    SerAlaLeuAlaValValLeuIlePheThrIleValValAspValLeu    354045    GlyAsnIleLeuValIleLeuSerValLeuArgAsnLysLysLeuGln    505560    AsnAlaGlyAsnLeuPheValValSerLeuSerIleAlaAspLeuVal    65707580    ValAlaValTyrProTyrProValIleLeuIleAlaIlePheGlnAsn    859095    GlyTrpThrLeuGlyAsnIleHisCysGlnIleSerGlyPheLeuMet    100105110    GlyLeuSerValIleGlySerValPheAsnIleThrAlaIleAlaIle    115120125    AsnArgTyrCysTyrIleCysHisSerLeuArgTyrAspLysLeuTyr    130135140    AsnGlnArgSerThrTrpCysTyrLeuGlyLeuThrTrpIleLeuThr    145150155160    IleIleAlaIleValProAsnPhePheValGlySerLeuGlnTyrAsp    165170175    ProArgIlePheSerCysThrPheAlaGlnThrValSerSerSerTyr    180185190    ThrIleThrValValValValHisPheIleValProLeuSerValVal    195200205    ThrPheCysTyrLeuArgIleTrpValLeuValIleGlnValLysHis    210215220    ArgValArgGlnAspPheLysGlnLysLeuThrGlnThrAspLeuArg    225230235240    AsnPheLeuThrMetPheValValPheValLeuPheAlaValCysTrp    245250255    AlaProLeuAsnPheIleGlyLeuAlaValAlaIleAsnProPheHis    260265270    ValAlaProLysIleProGluTrpLeuPheValLeuSerTyrPheMet    275280285    AlaTyrPheAsnSerCysLeuAsnAlaValIleTyrGlyValLeuAsn    290295300    GlnAsnPheArgLysGluTyrLysArgIleLeuMetSerLeuLeuThr    305310315320    ProArgLeuLeuPheLeuAspThrSerArgGlyGlyThrGluGlyLeu    325330335    LysSerLysProSerProAlaValThrAsnAsnAsnGlnAlaAspMet    340345350    LeuGlyGluAlaArgSerLeuTrpLeuSerArgArgAsnGlyAlaLys    355360365    MetValIleIleIleArgProArgLysAlaGlnIleAlaIleIleHis    370375380    GlnIlePheTrpProGlnSerSerTrpAlaThrCysArgGlnAspThr    385390395400    LysIleThrGlyGluGluAspGlyCysArgGluLeuCysLysAspGly    405410415    IleSerGlnArg    420    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1149 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: Coding Sequence    (B) LOCATION: 49...1146    (D) OTHER INFORMATION:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GGGAGCTCGACGCTCTGGGGATCCACCGGCGCCGGCCCTGCCAGCGCGATGGCGGGG57    MetAlaGly    CGGCTGTGGGGCTCGCCGGGCGGGACCCCCAAGGGCAACGGCAGCAGC105    ArgLeuTrpGlySerProGlyGlyThrProLysGlyAsnGlySerSer    51015    GCGCTGCTCAACGTCTCGCAGGCGGCGCCCGGCGCCGGGGACGGTGTG153    AlaLeuLeuAsnValSerGlnAlaAlaProGlyAlaGlyAspGlyVal    20253035    CGGCCGCGGCCCTCGTGGCTGGCCGCCACCCTCGCCTCCATCCTCATC201    ArgProArgProSerTrpLeuAlaAlaThrLeuAlaSerIleLeuIle    404550    TTCACCATCGTGGTGGACATCGTGGGCAACCTCCTGGTGGTCCTGTCC249    PheThrIleValValAspIleValGlyAsnLeuLeuValValLeuSer    556065    GTGTATCGGAACAAGAAGCTGAGGAACGCAGGGAATGTGTTTGTGGTG297    ValTyrArgAsnLysLysLeuArgAsnAlaGlyAsnValPheValVal    707580    AGCCTGGCAGTTGCAGACCTGCTGGTGGCCGTGTATCCGTACCCCTTG345    SerLeuAlaValAlaAspLeuLeuValAlaValTyrProTyrProLeu    859095    GCGCTGGCGTCTATAGTTAACAATGGGTGGAGCCTGAGCTCCCTGCAT393    AlaLeuAlaSerIleValAsnAsnGlyTrpSerLeuSerSerLeuHis    100105110115    TGCCAACTTAGTGGCTTCCTGATGGGCTTGAGCGTCATCGGGTCCGTT441    CysGlnLeuSerGlyPheLeuMetGlyLeuSerValIleGlySerVal    120125130    TTCAGCATCACGGGAATTGCCATCAACCGCTATTGCTGCATCTGCCAC489    PheSerIleThrGlyIleAlaIleAsnArgTyrCysCysIleCysHis    135140145    AGCCTCAGATACGGCAAGCTGTATAGCGGCACGAATTCCCTCTGCTAC537    SerLeuArgTyrGlyLysLeuTyrSerGlyThrAsnSerLeuCysTyr    150155160    GTGTTCCTGATCTGGACGCTGACGCTCGTGGCGATCGTGCCCAACCTG585    ValPheLeuIleTrpThrLeuThrLeuValAlaIleValProAsnLeu    165170175    TGTGTGGGGACCCTGCAGTACGACCCGAGGATCTATTCCTGTACCTTC633    CysValGlyThrLeuGlnTyrAspProArgIleTyrSerCysThrPhe    180185190195    ACGCAGTCCGTCAGCTCAGCCTACACGATCGCCGTGGTGGTGTTCCAT681    ThrGlnSerValSerSerAlaTyrThrIleAlaValValValPheHis    200205210    TTCATAGTTCCGATGCTCGTAGTCGTCTTCTGTTACCTGAGAATCTGG729    PheIleValProMetLeuValValValPheCysTyrLeuArgIleTrp    215220225    GCCCTGGTTCTTCAGGTCAGATGGAAGGTGAAACCGGACAACAAACCG777    AlaLeuValLeuGlnValArgTrpLysValLysProAspAsnLysPro    230235240    AAACTGAAGCCCCAGGACTTCAGGAATTTTGTCACCATGTTTGTGGTT825    LysLeuLysProGlnAspPheArgAsnPheValThrMetPheValVal    245250255    TTTGTCCTCTTTGCCATTTGCTGGGCTCCTCTGAACTTCATTGGTCTC873    PheValLeuPheAlaIleCysTrpAlaProLeuAsnPheIleGlyLeu    260265270275    GTTGTGGCCTCGGACCCCGCCAGCATGGCACCCAGGATCCCCGAGTGG921    ValValAlaSerAspProAlaSerMetAlaProArgIleProGluTrp    280285290    CTGTTTGTGGCTAGTTACTATATGGCATATTTCAACAGCTGCCTCAAT969    LeuPheValAlaSerTyrTyrMetAlaTyrPheAsnSerCysLeuAsn    295300305    GCGATCATATATGGACTACTGAACCAAAATTTCAGGCAGGAATACAGA1017    AlaIleIleTyrGlyLeuLeuAsnGlnAsnPheArgGlnGluTyrArg    310315320    AAAATTATAGTCTCATTGTGTACCACCAAGATGTTCTTTGTGGATAGC1065    LysIleIleValSerLeuCysThrThrLysMetPhePheValAspSer    325330335    TCCAATCATGTAGCAGATAGAATTAAACGCAAACCCTCTCCATTAATA1113    SerAsnHisValAlaAspArgIleLysArgLysProSerProLeuIle    340345350355    GCCAACCATAACCTAATAAAGGTGGACTCCGTTTAA1149    AlaAsnHisAsnLeuIleLysValAspSerVal    360365    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 366 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetAlaGlyArgLeuTrpGlySerProGlyGlyThrProLysGlyAsn    151015    GlySerSerAlaLeuLeuAsnValSerGlnAlaAlaProGlyAlaGly    202530    AspGlyValArgProArgProSerTrpLeuAlaAlaThrLeuAlaSer    354045    IleLeuIlePheThrIleValValAspIleValGlyAsnLeuLeuVal    505560    ValLeuSerValTyrArgAsnLysLysLeuArgAsnAlaGlyAsnVal    65707580    PheValValSerLeuAlaValAlaAspLeuLeuValAlaValTyrPro    859095    TyrProLeuAlaLeuAlaSerIleValAsnAsnGlyTrpSerLeuSer    100105110    SerLeuHisCysGlnLeuSerGlyPheLeuMetGlyLeuSerValIle    115120125    GlySerValPheSerIleThrGlyIleAlaIleAsnArgTyrCysCys    130135140    IleCysHisSerLeuArgTyrGlyLysLeuTyrSerGlyThrAsnSer    145150155160    LeuCysTyrValPheLeuIleTrpThrLeuThrLeuValAlaIleVal    165170175    ProAsnLeuCysValGlyThrLeuGlnTyrAspProArgIleTyrSer    180185190    CysThrPheThrGlnSerValSerSerAlaTyrThrIleAlaValVal    195200205    ValPheHisPheIleValProMetLeuValValValPheCysTyrLeu    210215220    ArgIleTrpAlaLeuValLeuGlnValArgTrpLysValLysProAsp    225230235240    AsnLysProLysLeuLysProGlnAspPheArgAsnPheValThrMet    245250255    PheValValPheValLeuPheAlaIleCysTrpAlaProLeuAsnPhe    260265270    IleGlyLeuValValAlaSerAspProAlaSerMetAlaProArgIle    275280285    ProGluTrpLeuPheValAlaSerTyrTyrMetAlaTyrPheAsnSer    290295300    CysLeuAsnAlaIleIleTyrGlyLeuLeuAsnGlnAsnPheArgGln    305310315320    GluTyrArgLysIleIleValSerLeuCysThrThrLysMetPhePhe    325330335    ValAspSerSerAsnHisValAlaAspArgIleLysArgLysProSer    340345350    ProLeuIleAlaAsnHisAsnLeuIleLysValAspSerVal    355360365    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 867 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: Coding Sequence    (B) LOCATION: 1...864    (D) OTHER INFORMATION:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GGAAACATCTTTGTGGTGAGCTTAGCGGTGGCAGACCTGGTGGTGGCC48    GlyAsnIlePheValValSerLeuAlaValAlaAspLeuValValAla    151015    ATTTATCCGTACCCGTTGGTGCTGATGTCGATATTTAACAACGGGTGG96    IleTyrProTyrProLeuValLeuMetSerIlePheAsnAsnGlyTrp    202530    AACCTGGGCTATCTGCACTGCCAAGTCAGTGGGTTCCTGATGGGCCTG144    AsnLeuGlyTyrLeuHisCysGlnValSerGlyPheLeuMetGlyLeu    354045    AGCGTCATCGGCTCCATATTCAACATCACCGGCATCGCCATCAACCGC192    SerValIleGlySerIlePheAsnIleThrGlyIleAlaIleAsnArg    505560    TACTGTTACATCTGCCACAGTCTCAAGTGCGACAAACTGTACAGCAGC240    TyrCysTyrIleCysHisSerLeuLysCysAspLysLeuTyrSerSer    65707580    AAGAACTCCCTCTGCTACGTGCTCCTCATATGGCTCCTGACGGCGGCC288    LysAsnSerLeuCysTyrValLeuLeuIleTrpLeuLeuThrAlaAla    859095    GTCCTGCCCAACCTCCGTCGTGGGACTCTCCAGTACGAGCCGAGGATC336    ValLeuProAsnLeuArgArgGlyThrLeuGlnTyrGluProArgIle    100105110    TACTCGTGCACCTTCGCCCAGTCCGTCAGCTCCGCCTACACCATCGCC384    TyrSerCysThrPheAlaGlnSerValSerSerAlaTyrThrIleAla    115120125    GTGGTGGTTTTCCACTTCCTCGTCCCCATGATCATAGTCATCTTCTGT432    ValValValPheHisPheLeuValProMetIleIleValIlePheCys    130135140    TACCTGAGAATATGGATCCTGGTTCTCCAGGTCAGACAGAGGGTGAAA480    TyrLeuArgIleTrpIleLeuValLeuGlnValArgGlnArgValLys    145150155160    CCTGACCGCAAACCCAAACTGAAACCACACGACTTCAGGAATTTTGTC528    ProAspArgLysProLysLeuLysProHisAspPheArgAsnPheVal    165170175    ACCATGTTTGTGGTTTTTGTCCTTTTTGCCATTTGCTGGGCTCCTCTG576    ThrMetPheValValPheValLeuPheAlaIleCysTrpAlaProLeu    180185190    AACTTCATTGGCCTGGCCGTGGCCTCTGACCCCGCCAGCATGGTGCCT624    AsnPheIleGlyLeuAlaValAlaSerAspProAlaSerMetValPro    195200205    AGGATCCCAGAGTGGCTGTTTGTGGCCAGTTACTACATGGCGTATTTC672    ArgIleProGluTrpLeuPheValAlaSerTyrTyrMetAlaTyrPhe    210215220    AACAGCTGCCTCAATGCCATTATATCGGGCTACTGGAACCAAAATTTC720    AsnSerCysLeuAsnAlaIleIleSerGlyTyrTrpAsnGlnAsnPhe    225230235240    AGGAAGGAATACAGGAGAATTATAGTCTCGCTCGTGACAGCCAGGGTG768    ArgLysGluTyrArgArgIleIleValSerLeuValThrAlaArgVal    245250255    TTCTTTGTGGACAGCTCTAACGACGTGGCCGATAGGGTTAAATGGAAA816    PhePheValAspSerSerAsnAspValAlaAspArgValLysTrpLys    260265270    CCGTCTCCACTGATGACCAACAATAATGTAGTAAAGGTGGACTCCGTT864    ProSerProLeuMetThrAsnAsnAsnValValLysValAspSerVal    275280285    TAA867    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 288 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GlyAsnIlePheValValSerLeuAlaValAlaAspLeuValValAla    151015    IleTyrProTyrProLeuValLeuMetSerIlePheAsnAsnGlyTrp    202530    AsnLeuGlyTyrLeuHisCysGlnValSerGlyPheLeuMetGlyLeu    354045    SerValIleGlySerIlePheAsnIleThrGlyIleAlaIleAsnArg    505560    TyrCysTyrIleCysHisSerLeuLysCysAspLysLeuTyrSerSer    65707580    LysAsnSerLeuCysTyrValLeuLeuIleTrpLeuLeuThrAlaAla    859095    ValLeuProAsnLeuArgArgGlyThrLeuGlnTyrGluProArgIle    100105110    TyrSerCysThrPheAlaGlnSerValSerSerAlaTyrThrIleAla    115120125    ValValValPheHisPheLeuValProMetIleIleValIlePheCys    130135140    TyrLeuArgIleTrpIleLeuValLeuGlnValArgGlnArgValLys    145150155160    ProAspArgLysProLysLeuLysProHisAspPheArgAsnPheVal    165170175    ThrMetPheValValPheValLeuPheAlaIleCysTrpAlaProLeu    180185190    AsnPheIleGlyLeuAlaValAlaSerAspProAlaSerMetValPro    195200205    ArgIleProGluTrpLeuPheValAlaSerTyrTyrMetAlaTyrPhe    210215220    AsnSerCysLeuAsnAlaIleIleSerGlyTyrTrpAsnGlnAsnPhe    225230235240    ArgLysGluTyrArgArgIleIleValSerLeuValThrAlaArgVal    245250255    PhePheValAspSerSerAsnAspValAlaAspArgValLysTrpLys    260265270    ProSerProLeuMetThrAsnAsnAsnValValLysValAspSerVal    275280285    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    AsnProXaaXaaTyr    15    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    AlaIleAlaIleAsnArgTyr    15    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    PheAlaValCysTrpAlaProLeu    15    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GlyAsnXaaPheValVal    15    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1085 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: Coding Sequence    (B) LOCATION: 33...1082    (D) OTHER INFORMATION:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    ATGGCCCTGCGGCCGGGACGCGAACAGGGACCATGCAGGGCAACGGCAGCGCG53    MetGlnGlyAsnGlySerAla    15    CTGCCCAACGCCTCCCAGCCCGTGCTCCGCGGGGACGGCGCGCGGCCC101    LeuProAsnAlaSerGlnProValLeuArgGlyAspGlyAlaArgPro    101520    TCGTGGCTGGCGTCCGCCCTAGCCTGCGTCCTCATCTTCACCATCGTG149    SerTrpLeuAlaSerAlaLeuAlaCysValLeuIlePheThrIleVal    253035    GTGGACATCCTGGGCAACCTCCTGGTCATCCTGTCGGTGTATCGGAAC197    ValAspIleLeuGlyAsnLeuLeuValIleLeuSerValTyrArgAsn    40455055    AAGAAGCTCAGGAACGCAGGAAACATCTTTGTGGTGAGCTTAGCGGTG245    LysLysLeuArgAsnAlaGlyAsnIlePheValValSerLeuAlaVal    606570    GCAGACCTGGTGGTGGCCATTTATCCGTACCCGTTGGTGCTGATGTCG293    AlaAspLeuValValAlaIleTyrProTyrProLeuValLeuMetSer    758085    ATATTTAACAACGGGTGGAACCTGGGCTATCTGCACTGCCAAGTCAGT341    IlePheAsnAsnGlyTrpAsnLeuGlyTyrLeuHisCysGlnValSer    9095100    GGGTTCCTGATGGGCCTGAGCGTCATCGGCTCCATATTCAACATCACC389    GlyPheLeuMetGlyLeuSerValIleGlySerIlePheAsnIleThr    105110115    GGCATCGCCATCAACCGCTACTGCTACATCTGCCACAGTCTCAAGTAC437    GlyIleAlaIleAsnArgTyrCysTyrIleCysHisSerLeuLysTyr    120125130135    GACAAACTGTACAGCAGCAAGAACTCCCTCTGCTACGTGCTCCTCATA485    AspLysLeuTyrSerSerLysAsnSerLeuCysTyrValLeuLeuIle    140145150    TGGCTCCTGACGCTGGCGGCCGTCCTGCCCAACCTCCGTGCAGGGACT533    TrpLeuLeuThrLeuAlaAlaValLeuProAsnLeuArgAlaGlyThr    155160165    CTCCAGTACGACCCGAGGATCTACTCGTGCACCTTCGCCCAGTCCGTC581    LeuGlnTyrAspProArgIleTyrSerCysThrPheAlaGlnSerVal    170175180    AGCTCCGCCTACACCATCGCCGTGGTGGTTTTCCACTTCCTCGTCCCC629    SerSerAlaTyrThrIleAlaValValValPheHisPheLeuValPro    185190195    ATGATCATAGTCATCTTCTGTTACCTGAGAATATGGATCCTGGTTCTC677    MetIleIleValIlePheCysTyrLeuArgIleTrpIleLeuValLeu    200205210215    CAGGTCAGACAGAGGGTGAAACCTGACCGCAAACCCAAACTGAAACCA725    GlnValArgGlnArgValLysProAspArgLysProLysLeuLysPro    220225230    CAGGACTTCAGGAATTTTGTCACCATGTTTGTGGTTTTTGTCCTCTTT773    GlnAspPheArgAsnPheValThrMetPheValValPheValLeuPhe    235240245    GCCATTTGCTGGGCTCCTCTGAACTTCATTGGCCTGGCCGTGGCCTCT821    AlaIleCysTrpAlaProLeuAsnPheIleGlyLeuAlaValAlaSer    250255260    GACCCCGCCAGCATGGTGCCTAGGATCCCAGAGTGGCTGTTTGTGGCC869    AspProAlaSerMetValProArgIleProGluTrpLeuPheValAla    265270275    AGTTACTACATGGCGTATTTCAACAGCTGCCTCAATGCCATTATATAC917    SerTyrTyrMetAlaTyrPheAsnSerCysLeuAsnAlaIleIleTyr    280285290295    GGGCTACTGAACCAAAATTTCAGGAAGGAATACAGGAGAATTATAGTC965    GlyLeuLeuAsnGlnAsnPheArgLysGluTyrArgArgIleIleVal    300305310    TCGCTCTGTACAGCCAGGGTGTTCTTTGTGGACAGCTCTAACGACGTG1013    SerLeuCysThrAlaArgValPhePheValAspSerSerAsnAspVal    315320325    GCCGATAGGGTTAAATGGAAACCGTCTCCACTGATGACCAACAATAAT1061    AlaAspArgValLysTrpLysProSerProLeuMetThrAsnAsnAsn    330335340    GTAGTAAAGGTGGACTCCGTTTAA1085    ValValLysValAspSerVal    345350    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 350 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    MetGlnGlyAsnGlySerAlaLeuProAsnAlaSerGlnProValLeu    151015    ArgGlyAspGlyAlaArgProSerTrpLeuAlaSerAlaLeuAlaCys    202530    ValLeuIlePheThrIleValValAspIleLeuGlyAsnLeuLeuVal    354045    IleLeuSerValTyrArgAsnLysLysLeuArgAsnAlaGlyAsnIle    505560    PheValValSerLeuAlaValAlaAspLeuValValAlaIleTyrPro    65707580    TyrProLeuValLeuMetSerIlePheAsnAsnGlyTrpAsnLeuGly    859095    TyrLeuHisCysGlnValSerGlyPheLeuMetGlyLeuSerValIle    100105110    GlySerIlePheAsnIleThrGlyIleAlaIleAsnArgTyrCysTyr    115120125    IleCysHisSerLeuLysTyrAspLysLeuTyrSerSerLysAsnSer    130135140    LeuCysTyrValLeuLeuIleTrpLeuLeuThrLeuAlaAlaValLeu    145150155160    ProAsnLeuArgAlaGlyThrLeuGlnTyrAspProArgIleTyrSer    165170175    CysThrPheAlaGlnSerValSerSerAlaTyrThrIleAlaValVal    180185190    ValPheHisPheLeuValProMetIleIleValIlePheCysTyrLeu    195200205    ArgIleTrpIleLeuValLeuGlnValArgGlnArgValLysProAsp    210215220    ArgLysProLysLeuLysProGlnAspPheArgAsnPheValThrMet    225230235240    PheValValPheValLeuPheAlaIleCysTrpAlaProLeuAsnPhe    245250255    IleGlyLeuAlaValAlaSerAspProAlaSerMetValProArgIle    260265270    ProGluTrpLeuPheValAlaSerTyrTyrMetAlaTyrPheAsnSer    275280285    CysLeuAsnAlaIleIleTyrGlyLeuLeuAsnGlnAsnPheArgLys    290295300    GluTyrArgArgIleIleValSerLeuCysThrAlaArgValPhePhe    305310315320    ValAspSerSerAsnAspValAlaAspArgValLysTrpLysProSer    325330335    ProLeuMetThrAsnAsnAsnValValLysValAspSerVal    340345350    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1062 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: Coding Sequence    (B) LOCATION: 1...1059    (D) OTHER INFORMATION:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    ATGAAGGGCAATGTCAGCGAGCTGCTCAATGCCACTCAGCAGGCTCCA48    MetLysGlyAsnValSerGluLeuLeuAsnAlaThrGlnGlnAlaPro    151015    GGCGGCGGGGAGGGAGGGAGACCACGACCGTCCTGGATGGCCTCTACA96    GlyGlyGlyGluGlyGlyArgProArgProSerTrpMetAlaSerThr    202530    CTGGCCTTCATCCTCATCTTTACCATCGTGGTGGACATTCTGGGCAAC144    LeuAlaPheIleLeuIlePheThrIleValValAspIleLeuGlyAsn    354045    CTGCTGGTCATCCTGTCTGTGTACCGCAACAAGAAGCTCAGGAACTCA192    LeuLeuValIleLeuSerValTyrArgAsnLysLysLeuArgAsnSer    505560    GGGAATATATTTGTGGTGAGTTTAGCTGTGGCAGACCTCGTGGTGGCT240    GlyAsnIlePheValValSerLeuAlaValAlaAspLeuValValAla    65707580    GTTTACCCTTATCCCTTGGTGCTGACATCTATCCTTAACAACGGATGG288    ValTyrProTyrProLeuValLeuThrSerIleLeuAsnAsnGlyTrp    859095    AATCTGGGATATCTACACTGTCAAGTCAGCGCATTTCTAATGGGCTTG336    AsnLeuGlyTyrLeuHisCysGlnValSerAlaPheLeuMetGlyLeu    100105110    AGTGTCATCGGCTCGATATTGAACATCACGGGGATCGCTATGAACCGT384    SerValIleGlySerIleLeuAsnIleThrGlyIleAlaMetAsnArg    115120125    TACTGCTACATTTGCCACAGCCTCAAGTACGACAAAATATACAGTAAC432    TyrCysTyrIleCysHisSerLeuLysTyrAspLysIleTyrSerAsn    130135140    AAGAACTCGCTCTGCTACGTGTTCCTGATATGGATGCTGACACTCATC480    LysAsnSerLeuCysTyrValPheLeuIleTrpMetLeuThrLeuIle    145150155160    GCCATCATGCCCAACCTGCAAACCGGAACACTCCAGTACGATCCCCGG528    AlaIleMetProAsnLeuGlnThrGlyThrLeuGlnTyrAspProArg    165170175    ATCTACTCCTGTACCTTCACCCAGTCTGTCAGCTCAGCGTACACGATA576    IleTyrSerCysThrPheThrGlnSerValSerSerAlaTyrThrIle    180185190    GCAGTGGTGGTTTTCCATTTCATCGTGCCTATGATTATTGTCATCTTC624    AlaValValValPheHisPheIleValProMetIleIleValIlePhe    195200205    TGCTACTTAAGGATATGGGTCCTGGTCCTTCAGGTCAGACGGAGGGTG672    CysTyrLeuArgIleTrpValLeuValLeuGlnValArgArgArgVal    210215220    AAACCCGACAACAAGCCCAAACTGAAGCCCCAGGACTTCAGGAACTTT720    LysProAspAsnLysProLysLeuLysProGlnAspPheArgAsnPhe    225230235240    GTCACCATGTTCGTAGTTTTTGTACTTTTTGCCATTTGTTGGGCCCCA768    ValThrMetPheValValPheValLeuPheAlaIleCysTrpAlaPro    245250255    CTCAACCTCATAGGTCTTATTGTGGCCTCAGACCCTGCCACCATGGTC816    LeuAsnLeuIleGlyLeuIleValAlaSerAspProAlaThrMetVal    260265270    CCCAGGATCCCAGAGTGGCTGTTCGTGGCTAGTTACTACCTGGCGTAC864    ProArgIleProGluTrpLeuPheValAlaSerTyrTyrLeuAlaTyr    275280285    TTCAACAGCTGCCTCAACGCAATTATATACGGACTACTGAATCAGAAT912    PheAsnSerCysLeuAsnAlaIleIleTyrGlyLeuLeuAsnGlnAsn    290295300    TTCAGAAAGGAATACAAAAAGATTATTGTCTCGTTGTGCACAGCCAAG960    PheArgLysGluTyrLysLysIleIleValSerLeuCysThrAlaLys    305310315320    ATGTTCTTTGTGGAGAGTTCAAATGAAGAAGCAGATAAGATTAAATGT1008    MetPhePheValGluSerSerAsnGluGluAlaAspLysIleLysCys    325330335    AAGCCCTCTCCACTAATACCCAATAATAACTTCCTCCCGGTGGACTCT1056    LysProSerProLeuIleProAsnAsnAsnPheLeuProValAspSer    340345350    GTTTAA1062    Val    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 353 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    MetLysGlyAsnValSerGluLeuLeuAsnAlaThrGlnGlnAlaPro    151015    GlyGlyGlyGluGlyGlyArgProArgProSerTrpMetAlaSerThr    202530    LeuAlaPheIleLeuIlePheThrIleValValAspIleLeuGlyAsn    354045    LeuLeuValIleLeuSerValTyrArgAsnLysLysLeuArgAsnSer    505560    GlyAsnIlePheValValSerLeuAlaValAlaAspLeuValValAla    65707580    ValTyrProTyrProLeuValLeuThrSerIleLeuAsnAsnGlyTrp    859095    AsnLeuGlyTyrLeuHisCysGlnValSerAlaPheLeuMetGlyLeu    100105110    SerValIleGlySerIleLeuAsnIleThrGlyIleAlaMetAsnArg    115120125    TyrCysTyrIleCysHisSerLeuLysTyrAspLysIleTyrSerAsn    130135140    LysAsnSerLeuCysTyrValPheLeuIleTrpMetLeuThrLeuIle    145150155160    AlaIleMetProAsnLeuGlnThrGlyThrLeuGlnTyrAspProArg    165170175    IleTyrSerCysThrPheThrGlnSerValSerSerAlaTyrThrIle    180185190    AlaValValValPheHisPheIleValProMetIleIleValIlePhe    195200205    CysTyrLeuArgIleTrpValLeuValLeuGlnValArgArgArgVal    210215220    LysProAspAsnLysProLysLeuLysProGlnAspPheArgAsnPhe    225230235240    ValThrMetPheValValPheValLeuPheAlaIleCysTrpAlaPro    245250255    LeuAsnLeuIleGlyLeuIleValAlaSerAspProAlaThrMetVal    260265270    ProArgIleProGluTrpLeuPheValAlaSerTyrTyrLeuAlaTyr    275280285    PheAsnSerCysLeuAsnAlaIleIleTyrGlyLeuLeuAsnGlnAsn    290295300    PheArgLysGluTyrLysLysIleIleValSerLeuCysThrAlaLys    305310315320    MetPhePheValGluSerSerAsnGluGluAlaAspLysIleLysCys    325330335    LysProSerProLeuIleProAsnAsnAsnPheLeuProValAspSer    340345350    Val    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1105 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: Coding Sequence    (B) LOCATION: 13...1098    (D) OTHER INFORMATION:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    GGAGAGTCTGCGATGTCAGAGAACGGCTCCTTCGCCAACTGCTGCGAGGCG51    MetSerGluAsnGlySerPheAlaAsnCysCysGluAla    1510    GGCGGGTGGGCAGTGCGCCCGGGCTGGTCGGGGGCTGGCAGCGCGCGG99    GlyGlyTrpAlaValArgProGlyTrpSerGlyAlaGlySerAlaArg    152025    CCCTCCAGGACCCCTCGACCTCCCTGGGTGGCTCCAGCGCTGTCCGCG147    ProSerArgThrProArgProProTrpValAlaProAlaLeuSerAla    30354045    GTGCTCATCGTCACCACCGCCGTGGACGTCGTGGGCAACCTCCTGGTG195    ValLeuIleValThrThrAlaValAspValValGlyAsnLeuLeuVal    505560    ATCCTCTCCGTGCTCAGGAACCGCAAGCTCCGGAACGCAGGTAATTTG243    IleLeuSerValLeuArgAsnArgLysLeuArgAsnAlaGlyAsnLeu    657075    TTCTTGGTGAGTCTGGCATTGGCTGACCTGGTGGTGGCCTTCTACCCC291    PheLeuValSerLeuAlaLeuAlaAspLeuValValAlaPheTyrPro    808590    TACCCGCTAATCCTCGTGGCCATCTTCTATGACGGCTGGGCCCTGGGG339    TyrProLeuIleLeuValAlaIlePheTyrAspGlyTrpAlaLeuGly    95100105    GAGGAGCACTGCAAGGCCAGCGCCTTTGTGATGGGCCTGAGCGTCATC387    GluGluHisCysLysAlaSerAlaPheValMetGlyLeuSerValIle    110115120125    GGCTCTGTCTTCAATATCACTGCCATCGCCATTAACCGCTACTGCTAC435    GlySerValPheAsnIleThrAlaIleAlaIleAsnArgTyrCysTyr    130135140    ATCTGCCACAGCATGGCCTACCACCGAATCTACCGGCGCTGGCACACC483    IleCysHisSerMetAlaTyrHisArgIleTyrArgArgTrpHisThr    145150155    CCTCTGCACATCTGCCTCATCTGGCTCCTCACCGTGGTGGCCTTGCTG531    ProLeuHisIleCysLeuIleTrpLeuLeuThrValValAlaLeuLeu    160165170    CCCAACTTCTTTGTGGGGTCCCTGGAGTACGACCCACGCATCTATTCC579    ProAsnPhePheValGlySerLeuGluTyrAspProArgIleTyrSer    175180185    TGCACCTTCATCCAGACCGCCAGCACCCAGTACACGGCGGCAGTGGTG627    CysThrPheIleGlnThrAlaSerThrGlnTyrThrAlaAlaValVal    190195200205    GTCATCCACTTCCTCCTCCCTATCGCTGTCGTGTCCTTCTGCTACCTG675    ValIleHisPheLeuLeuProIleAlaValValSerPheCysTyrLeu    210215220    CGCATCTGGGTGCTGGTGCTTCAGGCCCGCAGGAAAGCCAAGCCAGAG723    ArgIleTrpValLeuValLeuGlnAlaArgArgLysAlaLysProGlu    225230235    AGCAGGCTGTGCCTGAAGCCCAGCGACTTGCGGAGCTTTCTAACCATG771    SerArgLeuCysLeuLysProSerAspLeuArgSerPheLeuThrMet    240245250    TTTGTGGTGTTTGTGATCTTTGCCATCTGCTGGGCTCCACTTAACTGC819    PheValValPheValIlePheAlaIleCysTrpAlaProLeuAsnCys    255260265    ATCGGCCTCGCTGTGGCCATCAACCCCCAAGAAATGGCTCCCCAGATC867    IleGlyLeuAlaValAlaIleAsnProGlnGluMetAlaProGlnIle    270275280285    CCTGAGGGGCTATTTGTCACTAGCTACTTACTGGCTTATTTCAACAGC915    ProGluGlyLeuPheValThrSerTyrLeuLeuAlaTyrPheAsnSer    290295300    TGCCTGAATGCCATTGTCTATGGGCTCTTGAACCAAAACTTCCGCAGG963    CysLeuAsnAlaIleValTyrGlyLeuLeuAsnGlnAsnPheArgArg    305310315    GAATACAAGAGGATCCTCTTGGCCCTTTGGAACCCACGGCACTGCATT1011    GluTyrLysArgIleLeuLeuAlaLeuTrpAsnProArgHisCysIle    320325330    CAAGATGCTTCCAAGGGCAGCCACGCGGAGGGGCTGCAGAGCCCAGCT1059    GlnAspAlaSerLysGlySerHisAlaGluGlyLeuGlnSerProAla    335340345    CCACCCATCATTGGTGTGCAGCACCAGGCAGATGCTCTCTAGCCTG1105    ProProIleIleGlyValGlnHisGlnAlaAspAlaLeu    350355360    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 362 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    MetSerGluAsnGlySerPheAlaAsnCysCysGluAlaGlyGlyTrp    151015    AlaValArgProGlyTrpSerGlyAlaGlySerAlaArgProSerArg    202530    ThrProArgProProTrpValAlaProAlaLeuSerAlaValLeuIle    354045    ValThrThrAlaValAspValValGlyAsnLeuLeuValIleLeuSer    505560    ValLeuArgAsnArgLysLeuArgAsnAlaGlyAsnLeuPheLeuVal    65707580    SerLeuAlaLeuAlaAspLeuValValAlaPheTyrProTyrProLeu    859095    IleLeuValAlaIlePheTyrAspGlyTrpAlaLeuGlyGluGluHis    100105110    CysLysAlaSerAlaPheValMetGlyLeuSerValIleGlySerVal    115120125    PheAsnIleThrAlaIleAlaIleAsnArgTyrCysTyrIleCysHis    130135140    SerMetAlaTyrHisArgIleTyrArgArgTrpHisThrProLeuHis    145150155160    IleCysLeuIleTrpLeuLeuThrValValAlaLeuLeuProAsnPhe    165170175    PheValGlySerLeuGluTyrAspProArgIleTyrSerCysThrPhe    180185190    IleGlnThrAlaSerThrGlnTyrThrAlaAlaValValValIleHis    195200205    PheLeuLeuProIleAlaValValSerPheCysTyrLeuArgIleTrp    210215220    ValLeuValLeuGlnAlaArgArgLysAlaLysProGluSerArgLeu    225230235240    CysLeuLysProSerAspLeuArgSerPheLeuThrMetPheValVal    245250255    PheValIlePheAlaIleCysTrpAlaProLeuAsnCysIleGlyLeu    260265270    AlaValAlaIleAsnProGlnGluMetAlaProGlnIleProGluGly    275280285    LeuPheValThrSerTyrLeuLeuAlaTyrPheAsnSerCysLeuAsn    290295300    AlaIleValTyrGlyLeuLeuAsnGlnAsnPheArgArgGluTyrLys    305310315320    ArgIleLeuLeuAlaLeuTrpAsnProArgHisCysIleGlnAspAla    325330335    SerLysGlySerHisAlaGluGlyLeuGlnSerProAlaProProIle    340345350    IleGlyValGlnHisGlnAlaAspAlaLeu    355360    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    AsnAlaXaaXaaTyr    15    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    CysTyrIleCysHisSer    15    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    CTGTGCCTCTAAGAGCCACTTGGTTTC27    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    TCCTGGTGATCCTCTCCGTGCTCA24    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    AGCCAGATGAGGCAGATGTGCAGA24    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    TCCTGGTCATCCTGTCGGTGTATC24    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    CTGCTGTACAGTTTGTCGTACTTG24    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    GCAAGAGTGCGCCCTCTACTG21    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    GGCCTCACTTGCCTCCTGCAA21    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    CTAATCCTCGTGGCCAATCTTCTATG26    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    TTGGTGCTGATGTCGATATTTAACA25    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    CACTGAACTTCTGATTCGCAAACTT25    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    TATTGAAGACAGAGCCGATGACGCTCA27    __________________________________________________________________________

What is claimed is:
 1. Isolated DNA comprising the coding sequence ofSEQ ID NO:1, or a degenerate variant thereof, and encoding the aminoacid sequence of (SEQ ID No.2).
 2. Isolated DNA comprising the codingsequence of SEQ ID NO:3, or a degenerate variant thereof, and encodingthe amino acid sequence of SEQ ID NO:4.
 3. Isolated DNA comprising thecoding sequence of SEQ ID NO:5, or a degenerate variant thereof, andencoding an amino acid sequence comprising the amino acid sequence ofSEQ ID NO.6.
 4. Isolated DNA comprising the coding sequence of SEQ IDNO:11, or a degenerate variant thereof, and encoding an amino acidsequence comprising the amino acid sequence of SEQ ID NO:12.
 5. IsolatedDNA comprising the coding sequence of SEQ ID NO:13, or a degeneratevariant thereof, and encoding an amino acid sequence comprising theamino acid sequence of SEQ ID NO:14.
 6. Isolated DNA comprising thecoding sequence of SEQ ID NO:15, or a degenerate variant thereof, andencoding an amino acid sequence comprising the amino acid sequence ofSEQ ID NO:16.
 7. Isolated DNA which hybridizes to the DNA sequence ofFIG. 1 (SEQ ID NO:1) under conditions of high stringency.
 8. IsolatedDNA which hybridizes to the DNA sequence of FIG. 2 (SEQ ID NO:3) underconditions of high stringency.
 9. Isolated DNA which hybridizes to theDNA sequence of FIG. 4 (SEQ ID NO:5) under conditions of highstringency.
 10. Isolated DNA which hybridizes to the DNA sequence ofFIG. 5 (SEQ ID NO:11) under conditions of high stringency.
 11. IsolatedDNA which hybridizes to the DNA sequence of FIG. 3 (SEQ ID NO:13) underconditions of high stringency.
 12. Isolated DNA which hybridizes to theDNA sequence of FIG. 6 (SEQ ID NO:15) under conditions of highstringency.
 13. A vector comprising the DNA of claim
 4. 14. A vectorcomprising the DNA of claim
 10. 15. A vector comprising the DNA of claim11.
 16. A vector comprising the DNA of claim
 12. 17. A cell whichcontains the DNA of claim
 10. 18. A cell which contains the DNA of claim11.
 19. A cell which contains the DNA of claim 12.